Immune Modulation By Regulating Expression Of The &#34;Minor&#34; Gene In Immune Dendritic Cells

ABSTRACT

Mitogen induced nuclear orphan receptor (MINOR) is described as inducing apoptosis in dendritic cells (DCs). Downregulation of its expression results in a downregulation of apoptosis. A novel approach of inhibiting DC apoptosis is described employing small interfering RNA (siRNA) that targets MINOR. Improved DC-based vaccines exhibiting longer lifespan of DCs, increased potency of DCs, and enhanced immunogenicity are described.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationSer. No. 60/542,987 and 60/561,417 filed Feb. 9, 2004 and Apr. 12, 2004,respectively, the entire disclosures of which are incorporated herein byreference in its entirety.

FIELD OF THE INVENTION

The present invention relates to the modulation of a targeted gene incertain immune cells. This modulation can result in the inhibition orstimulation of certain immune response processes.

BACKGROUND OF THE INVENTION

Dendritic cells (DCs) are scarce cells in the immune system that arespecialized in processing antigens, presenting to naive T lymphocytes,and playing an important role in initiating host immune responses. DCsalso play an important role in maintaining tolerance. Studies usinglabeled DCs suggest that they are replaced every 3-4 days(Asavaroengchai, W., Kotera, Y. & Mule, J. J., Proc Natl Acad Sci U S A99, 931-6, 2002), implying a limited window during which T cells canencounter DCs presenting antigens. While many advances have been made inunderstanding the nature of antigen processing and presentation,regulation of DC lifespan has not been well explored.

Apoptosis is a highly complex process that allows a cell to commitsuicide in a controlled manner. Death receptors belonging to the tumornecrosis factor receptor superfamily can transmit apoptotic signalsinitiated by their specific ligands. One central component of theapoptotic machinery is a family of proteases, the caspases, whichparticipate in a cascade of proteolytic cleavages, finally resulting inthe death of the cell. Of note, a number of reports have appeareddescribing a caspase-independent form of apoptosis.

Previous studies have shown that manipulation of the bcl-2 pathway ledto inhibition of apoptotic cell death of DCs. DCs were shown todownregulate the anti-apoptotic bcl-2 upon maturation, leading to theirprogression to cell death. Mice were generated that were transgenic forbcl-2, and that had increased numbers of DCs. Further, DCs from thesetransgenic mice as vaccines generated an enhanced immune response.(Nopora, A. & Brocker, T., J Immunol 169, 3006-14, 2002). Also, whiletumors can induce apoptosis of DCs, transduction of ex vivo-generatedDCs with the anti-apoptotic Bcl-xl increased their resistance toapoptosis and improved tumor vaccine efficacy. (Pirtskhalaishvili, G. etal., J Immunol 165, 1956-62, 2000)

Nurr77 (NGFI-γ or TR3), an orphan member of the steroid/thyroid/retinoidnuclear receptor superfamily (Kastner et al., 1995; Mangelsdorf andEvans, 1995; Zhang, 2002), plays roles in regulating growth andapoptosis (Winoto and Littman, 2002; Zamzami and Kroemer, 2001; Zhang2002) in T lymphocytes. The Nurr77 family members, Nur77, Nor-1, andNurr1, have the typical steroid receptor organization composed of anN-terminal transactivation domain, a central DNA-binding domaincontaining two zinc fingers, and a C-terminus with homology tohormone-binding domains (Carson et al., 1990). Recent studies indicatethat Nur77 translocates from the nucleus to the mitochondria, where itdirectly exerts its pro-apoptic effects.

The role of Nur77 in T cell development has been fairly well documented.The actual mechanism of cell death induction by these molecules is notas clear. Nur77 is a nuclear, DNA binding, transcriptional regulator, sothe most likely mechanism is via transcription of downstream effectors(Kuang, A. A., Cado, D. & Winoto, A., Eur J Immunol 29, 3722-8, 1999).However, other evidence suggests that Nur77 can mediate its apoptoticaffect in the mitochondria (Brenner, C. & Kroemer, G., Science 289,1150-1, 2000).

Mouse MINOR belongs to the Nur77 family of orphan receptors thatincludes Nur77 and the rat homologue of MINOR, Nor 1, which is involvedin activation induced cell death of T cells (Cheng, L. E., Chan, F. K.,Cado, D. & Winoto, A., Embo J 16, 1865-75, 1997; Liu, Z. G., Smith, S.W., McLaughlin, K. A., Schwartz, L. M. & Osborne, B. A., Nature 367,281-4, 1994). and also caspase-independent cell death of macrophages.(Kim, S. O., Ono, K., Tobias, P. S. & Han, J., J Exp. Med 197, 1441-52,2003). The role of Nur77 in T cell development has been fairly welldocumented. Interestingly, Nor-1 appears to be functionally redundantwith Nur77 not only in DNA binding specificity and ability totransactivate from the NBRE promoter, but also in its ability to induceapoptosis in T cells. Furthermore, a dominant negative Nur77 genefragment lacking the transactivation domain blocks the induction ofapoptosis by both Nur77 and Nor-1. In addition, transgenic miceexpressing the rat MINOR homologue, under the control of the lckpromoter, exhibited a 15-fold reduction in their thymocyte number,clearly showing the impact of this pathway on T cells (Cheng, L. E.,Chan, F. K., Cado, D. & Winoto, A., Embo J 16, 1865-75, 1997). However,the notion that Nor-1 has been shown to bind and activate transcriptionfrom the NBRE indicates that it is likely capable of activating the samegenes that are activated by other members of this orphan receptorfamily. Indeed, Nor-1 has been shown to express in activated T cells anddemonstrated for its role in T cell receptor-mediated apoptosis in vivo.

Improving vaccination strategies for tumors is a significant goal ofimmunotherapy. As a result of the potency of dendritic cells (DCs) asantigen presenting cells (APCs), DCs have been investigated for boththeir biology and potential as therapeutic agents. While many advanceshave occurred in this field, highly potent and durable anti-tumor immuneresponses have been difficult to achieve through these vaccines. It nowappears that DC vaccines can elicit strong immune responses, but theyare limited, in part, by their short lifespan in vivo. While muchemphasis has been placed on studying antigen (Ag) uptake, processing,and presentation, as well as co-stimulatory signal delivery by DCs,little is known about regulation of DC lifespan.

DC-based vaccines have been tested in a number of animal models, andhave also been translated to clinical medicine in several trials. Thus,the hope for successful immunotherapy through this mechanism is high. Toproduce DC vaccines, there are two primary methods: First, precursorcells can be isolated, grown ex vivo and differentiated in culture, thensubsequently re-infused; second, they can be generated in vivo throughthe systemic administration of GM-CSF and/or Flt-3 ligand (FLT3L). Whilethese approaches have led to some degree of tumor immunity, they havealso exhibited limitations (Borges, L. et al., J Immunol 163, 1289-97,1999). The identification of tumor-specific and tumor-associated Ags hasled to therapies such as vaccination with recombinant viruses or DCsmodified to express Ags, but these have also had limited effects.(Marshall, J. L. et al., J Clin Oncol 18, 3964-73, 2000; Morse, M. A. etal., Cancer Invest 21, 341-9, 2003; Ridgway, D., Cancer Invest 21,873-86, 2003).

Therapeutic cancer vaccination depends on effective transfer of Ag toDCs and trafficking of the DCs to the secondary lymphoid organs. Whilemany clinical trials have been initiated with ex vivo-generated DCs, forthe most part, no long-term cures have been achieved. Of note, DCs aremore resistant to some apoptotic pathways than other cells as a resultof expression of molecules such as FLICE inhibitory protein (cFLIP),which can block caspase 8 activation and the subsequent apoptoticcascade (Leverkus, M. et al., Blood 96, 2628-31, 2000). Additionally,signaling by TRANCE, or CD154 have been shown to prevent apoptosis inDCs (McLellan, A. et al., Eur J Immunol 30, 2612-9, 2000; Wong, B. R. etal., J Exp Med 186, 2075-80, 1997). In spite of these, the DCs generallyhave a very short lifespan in vivo. In fact, animal models and clinicaltrials suggest that one major issue with the ex vivo expansion andloading of DCs followed by re-injection is that relatively few DCssuccessfully traffic to spleen or lymph nodes (Eggert, A. A. et al.,Cancer Res 59, 3340-5, 1999), and those that do are rapidly cleared byhost CTL (Cayeux, S. et al., Eur J Immunol 29, 225-34, 1999). Inaddition, NK cells can kill DCs through TRAIL-mediated apoptosis(Hayakawa, Y. et al., J Immunol 172, 123-9, 2004), and further studiesusing labeled DCs suggest that the cells are replaced every 3-4 days(Kamath, A. T., Henri, S., Battye, F., Tough, D. F. & Shortman, K.,Blood 100, 1734-41, 2002), implying a limited window during which Tcells can encounter DCs presenting Ag. Consequently, it would be highlydesirable if methods were available to increase the lifespan of DCs andmaximize their effect in a number of immune processes involving Agpresentation, including vaccinations.

SUMMARY OF THE INVENTION

Recent investigation of some of the genes that are upregulated in DCshas led to the identification of one that appears to play a significantrole in limiting the lifespan of DCs, mitogen induced nuclear orphanreceptor (MINOR). The subtractive hybridization analysis betweenactivated macrophages and DCs that was employed to identify selectivelyexpressed genes revealed a highly upregulated expression of the mousehomolog to human MINOR in mature DCs.

Extending the longevity of DCs allows for their improved immunogenicity.In one embodiment, the present invention is directed to a method forsubstantially inhibiting apoptosis in dendritic cells comprising theprevention or inhibition of the expression of MINOR in said cells. Aninhibition of apoptosis constitutes an experimentally quantifiable levelof inhibition, as shown in the Examples, below. A prevention orinhibition of the expression of MINOR constitutes a downregulation ofthe expression of MINOR great enough to result in some measurable degreeof inhibition of apoptosis in the DCs.

In another embodiment of the invention, the expression of MINOR in DCsis prevented or inhibited by the transduction of the cells with alentiviral vector encoding an siRNA construct having substantialsequence homology to MINOR. Substantial sequence homology constitutesabout 60% or greater sequence homology, preferably about 75% or greater,more preferably about 85% or greater. The sequence homology must begreat enough to allow effective targeting of MINOR by the siRNAconstruct.

In yet another embodiment of the invention, the dendritic cells are bonemarrow dendritic cells.

In one embodiment, the invention is directed to a method for improvingthe survival time after infusion of ex vivo-generated dendritic cells,said method comprising transducing said cells with a lentiviral vectorencoding an siRNA construct having substantial sequence homology toMINOR or through additional means of introducing a MINOR-blockingreagent, e.g., with a plasmid construct through transfection by anynumber of standard methods, and infusing the transduced cells into amammalian subject. In a further embodiment of the invention, themammalian subject is human.

In one embodiment, the invention is directed to enhancing theimmunogenicity of dendritic cells, said method comprising transducingsaid cells with a lentiviral vector encoding an siRNA construct havingsubstantial sequence homology to MINOR.

In another embodiment, the invention is directed to a method forenhancing the capacity for dendritic cells to stimulate tolerant Tcells, said method comprising transducing said cells with a lentiviralvector encoding an siRNA construct having substantial sequence homologyto MINOR.

In one embodiment, the invention is directed to a dendritic cell-basedvaccine comprising siRNA having substantial sequence homology to MINOR.In another embodiment of the invention, the vaccine is for cancer, viraldisease, bacterial disease, or immune disorders.

In one embodiment, the invention is directed to a method for preparing aDC-based vaccine, comprising the step of preparing an siRNA constructhaving substantial sequence homology to MINOR to target MINOR on amolecular level.

In another embodiment, the invention is directed to a method ofpreserving the CD11c+population of dendritic cells, comprisingtransducing hematopoietic stem-progenitor cells with a lentiviral vectorencoding an siRNA construct having substantial sequence homology toMINOR.

In an additional embodiment, the invention is directed to a method forstably decreasing or substantially suppressing the expression of MINORin dendritic cells, said method comprising the steps of transducinghematopoietic stem-progenitor cells with a vector encoding an siRNAconstruct having substantial sequence homology to MINOR andtransplanting the transduced cells into a myeloablatively treatedmammalian subject.

Specifically in one embodiment of the present invention a dendritic cellis transduced with a construct containing the a small interfering RNAcomprising the double stranded nucleotide sequence of5′GATCCCCTGCCCTTGTCCGAGCTTTATTCAAGAGATAAAGCTCGGACAAGGGCATTTTTGGAAA-3′;forward and5′AGCTTTTCCAAAAATGCCCTTGTCCGAGCTTTATCTCTTGAATAAAGCTCGGACAAGGGCAGGG-3′;reverse.

In additional embodiments, the invention is directed to improved DCvaccines.

Thus, the present invention utilizes the discovery of MINOR as asignificant gene with regard to basic DC function in that it may helpregulate DC lifespan, thereby limiting uncontrolled T cell activationand also thereby serving as a target for improving DC-based therapies.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates the results of qPCR analysis of relative MINORexpression in vitro in DCs and activated macrophages; Lane 1 correspondsto activated macrophages; Lane 2 corresponds to day 4 cultures of bonemarrow DCs; Lane 3 corresponds to day 8 cultures of bone marrow DCs;Lane 4 corresponds to myeloid CD11c hi DCs isolated from Balb/c lymphnodes.

FIG. 2 schematically depicts the structure of MINOR and illustrates theresults of protein sequence alignment of MINOR with other members of theNur77/Nurr1 steroid hormone receptor family, showing % identity to themost similar sequences; the subfamilies of this gene are likewise shown.

FIG. 3 represents an immunoblot depicting MINOR expression in mouseBMDCs at the protein level.

FIG. 4 depicts the expression of MINOR and Nurr77 in various cell(primarily dendritic cell) populations, respectively, in bar graph form.

FIG. 5 illustrates cell death as measured via FACS analysis of theDC-like cell line DC 2.4 transfected with the rat homolog MINOR cDNA orpEGFP-N2. Cells were gated on GFP+ populations in order to specificallycompare transduced cells.

FIG. 6 illustrates in bar graph form the results of quantitative PCRanalysis of MINOR expression by bone marrow progenitor cell-derived DCstransduced with siRNA-MINOR (as compared with unmodified mature DCs).

FIG. 7 illustrates the FACS results of staining for apoptosis in DC2.4cells transduced with LV-siRNA-MINOR or LV-control-siRNA and thentransfected with rat Nor-1; transduced DC2.4 cells were gated based onGFP fluorescence (GFP+) and compared with the non-transduced DC2.4 (GFPnegative).

FIG. 8 illustrates the FACS results of staining for apoptosis in BMDCstransduced with LV-siRNA-MINOR-GFP or LI-control-siRNA-GFP. The FACSplots are gated either on GFP⁻ (right) or GFP+ (left) for eithersiRNA-MINOR or. control vs 7-AAD (indicating the dead population in theupper right quadrant); the GFP⁻ fractions in both populations representthe untransduced groups, while the GFP⁺ populations are transduced; thecontrol GFP⁺ populations express a control siRNA-GFP while the siRNA-GFPpopulation expresses both GFP and siRNA-MINOR.

FIG. 9 illustrates the results of monitoring the survival of ex vivogenerated BALB/c BMDCs transduced with siRNA-MINOR-GFP or control GFPand injected into mice; the histograms shown are gated on CD11c+ cellsfor this analysis.

FIG. 10 depicts, in bar graph, FACS plot, and point distribution graphform, T cell expansion, as measured in terms of staining for 6.5 andCD4, observed in mouse spleen and lymph nodes after transfer of 6.5BMDCs transduced with siRNA-MINOR-GFP or control-siRNA-GFP and pulsedwith class II restricted peptide for HA into the mice.

FIGS. 11 a and 11 b illustrate, in FACS plot and distribution graphform, respectively, the FACS results of staining with antibodies for thy1.2 and CD4 in ex vivo generated BMDCs expressing HA as self antigen(“142”), transduced with si-RNA MINOR or siRNA control and pulsed withHA class II peptide prior to subcutaneous injection into mice; unpulsedDCs were included as a control.

FIG. 12 illustrates, in bar graph form, the relative in vivo expression(in BALB.c mice) of MINOR upon systemic DC activation, in comparisonwith that measured for naïve DCs.

FIG. 13 illustrates, in bar graph form, the relative in vivo expressionof MINOR in mice transplanted with siRNA-MINOR- or siRNAcontrol-transduced HSCs and allowed to engraft; the lanes are asfollows: (1) plasmacytoid (p)DCs from the spleen of the 1203transplants; (2) control spleen pDCs; (3) LN pDCs from the 1203transplants, and (4) LN pDCs from the control transplants; values werenormalized to actin, and the lowest value was arbitrarily set to 1.

FIG. 14 illustrates the relative expression of siRNA-MINOR vs. controlsiRNA in CD11c+ cells vs. CD11c− cells; for the FACS analysis, the cellswere stained for CD11c and 7-AAD; shown are representative plots of LNfor GFP by CD11c, in order to compare relative expression of the vectorsin DC (upper 2 quads) vs non-DC (lower 2 quads) populations; the bargraph shows the average and SD of 6 mice/group.

FIG. 15 illustrates the FACS results for an analysis of expression ofsiRNA-MINOR vs. siRNA-control based on CD86 expression; shown arerepresentative FACS plots for siRNA-MINOR, left, and the control, right,for CD86 by GFP.

FIG. 16 illustrates the results of an analysis of the viability of theCD11c+ DC progeny of HSCs transduced with siRNA-MINOR vs. siRNA-controlprior to transplant; Separate comparisons were made within each group ofmice (control GFP⁺ vs control GFP⁻ and siRNA-MINOR GFP⁺ vs siRNA-MINORGFP⁺).

FIG. 17 illustrates the relative expression (normalized to actin) ofhuman MINOR in: CD34⁺ hematopoietic cell progenitors, LPSactivated-monocyte derived-macrophages, and activated dendritic cells.

FIG. 18 depicts the nucleotide sequence for MINOR (SEQ ID NO. 1).

DETAILED DESCRIPTION OF THE INVENTION

Improving vaccination strategies for tumors is a significant goal ofimmunotherapy. As a result of the potency of dendritic cells (DCs) asantigen presenting cells (APCs), DCs have been investigated for boththeir biology and potential as therapeutic agents. While many advanceshave occurred in this field, highly potent and durable anti-tumor immuneresponses have been difficult to achieve through these vaccines. It nowappears that DC vaccines can elicit strong immune responses, but theyare limited, in part by their short lifespans in vivo. While muchemphasis has been placed in studying antigen (Ag) uptake, processing andpresentation as well as costimulatory signal delivery by DCs, little isknown about regulation of DC lifespan. We have identified one gene thatappears to play a significant role in limiting the lifespan of DCs,mitogen induced nuclear orphan receptor (MINOR). The identification ofthis gene was made through a subtractive hybridization analysis betweenactivated macrophages and DCs, which revealed a highly upregulatedexpression of the mouse homolog to human MINOR in mature DCs. Manystudies have now focused on the features of DCs that allow them to besuch potent APCs, including analyses of signals for activation that areimportant for DC function (such as co-stimulatory molecules, moleculesinvolved in Ag processing and presentation, etc.). Once DCs have beenactivated, however, they are thought to have a relatively short lifespanin vivo, which presumably serves to limit clonal expansion in an immuneresponse. Thus, it appears that DCs are messengers with a limited timeto carry their Ag to secondary lymphoid organs and activate T cells inthe context of a pro-inflammatory environment.

Minor Gene and its Protein Product

MINOR was identified as a gene that is highly upregulated andselectively expressed in DCs. This gene belongs to the Nur77 family oforphan receptors that includes Nur77 and the rat homologue of MINOR, Nor1, which is involved in activation induced cell death of T cells (Cheng,L. E., Chan, F. K., Cado, D. & Winoto, A., Embo J 16, 1865-75, 1997;Liu, Z. G., Smith, S. W., McLaughlin, K. A., Schwartz, L. M. & Osborne,B. A., Nature 367, 281-4, 1994) and also caspase-independent cell deathof macrophages (Kim, S. O., Ono, K., Tobias, P. S. & Han, J., J. Exp.Med 197, 1441-52, 2003). The role of Nur77 in T cell development hasbeen fairly well documented. Interestingly, Nor-1, appears to befunctionally redundant with Nur77 not only in DNA binding specificityand ability to transactivate from the NBRE promoter, but also in itsability to induce apoptosis in T cells. Furthermore, a dominant negativeNur77 gene fragment lacking the transactivation domain blocks theinduction of apoptosis by both Nur77 and Nor-1. In addition, transgenicmice expressing the rat MINOR homologue, under the control of the lckpromoter, exhibited a 15 fold reduction in their thymocyte number,clearly showing the impact of this pathway on T cells (Cheng, L. E.,Chan, F. K., Cado, D. & Winoto, A., Embo J 16, 1865-75, 1997). Previousresults show that Nur77 is not upregulated as DCs mature thus we arehypothesizing that the mouse MINOR substitutes for the signal to induceapoptosis, in a selective fashion.

The actual mechanism of cell death induction by these molecules is notas clear. Nur77 is a nuclear, DNA binding, transcriptional regulator, sothe most likely mechanism is via transcription of downstream effectors(Kuang, A. A., Cado, D. & Winoto, A., Eur J Immunol 29, 3722-8, 1999).However, other evidence suggests that Nur77 can mediate its apoptoticeffect in the mitochondria (Brenner, C. & Kroemer, G., Science 289,1150-1, 2000). Using a GFP tagged Nur77 to follow its movement throughcells, a translocation from the nucleus to mitochondria was observedwith concomitant cytochrome c release in the presence of apoptosisinducing stimuli in these studies. The mitochondrial localization ofGFP-Nur77 and subsequent cytochrome c release was constitutive if thenuclear localization signals and DNA binding domain of this protein.Overexpression of a Nur77 dominant negative blocked both themitochondrial translocation of Nur77 and its induction of apoptosis (Li,H. et al., Science 289, 1159-64, 2000).

DC-Based Vaccines

One avenue of exploiting MINOR inhibition is through the potential forenhancement of DC vaccines. DC-based vaccines have been tested in anumber of animal models, and have also been translated to clinicalmedicine in several trials. To produce DC vaccines, there are twoprimary methods: First, precursor cells can be isolated, grown ex vivoand differentiated in culture, then subsequently re-infused; second theycan be generated in vivo through the systemic administration of GM-CSFand/or Flt-3 ligand (FLT3L). While these approaches have led to somedegree of tumor immunity, they have also had limitations (Borges, L. etal., J Immunol 163, 1289-97, 1999). The identification of tumor specificand tumor-associated Ags has led to therapies such as vaccination withrecombinant viruses or DCs modified to express Ags but these have alsohad limited effects. (Marshall, J. L. et al., J Clin Oncol 18, 3964-73,2000; Morse, M. A. et al., Cancer Invest 21, 341-9, 2003; Ridgway, D.,Cancer Invest 21, 873-86, 2003).

Therapeutic cancer vaccination depends on effective transfer of Ag toDCs and trafficking of the DCs to the secondary lymphoid organs. Whilemany clinical trials have been initiated with ex vivo generated DCs, forthe most part, no long term cures have been achieved. Interestingly, DCsare more resistant to some apoptotic pathways than other cells as aresult of expression of molecules such as FLICE inhibitory protein(cFLIP) which can block caspase 8 activation and the subsequentapoptotic cascade (Leverkus, M. et al., Blood 96, 2628-31, 2000).Additionally, signaling by TRANCE, or CD154 have been shown to preventapoptosis in DC (McLellan, A. et al., Eur J Immunol 30, 2612-9, 2000;Wong, B. R. et al., J Exp Med 186, 2075-80, 1997). In spite of these,they generally have a very short lifespan in vivo. In fact, animalmodels and clinical trials suggest that one major issue with ex vivoexpansion and loading of DCs followed by re-injection is that relativelyfew DCs successfully traffic to spleen or lymph nodes (Eggert, A. A. etal., Cancer Res 59, 3340-5, 1999) and those that do are rapidly clearedby host CTL (Cayeux, S. et al., Eur J Immunol 29, 225-34, 1999). Inaddition, NK cells can kill DCs through TRAIL mediated apoptosis(Hayakawa, Y. et al., J Immunol 172, 123-9, 2004), and further studiesusing labeled DCs suggest that they are replaced every 3-4 days (Kamath,A. T., Henri, S., Battye, F., Tough, D. F. & Shortman, K., Blood 100,1734-41, 2002), implying a limited window during which T cells canencounter DCs presenting Ag. Thus, extending the longevity of DCs wouldallow for their improved immunogenicity.

Enhancement of Ex Vivo DC Vaccines:

DC based therapies have been investigated for tumors for which theantigen is known, through antigen-specific activation, and also fortumors for which the antigen is not known, through whole tumorcell-based activation. The use of ex vivo DC vaccines has the potentialadvantage of Ag loading with multiple Ags, some of which are notidentified through the use of whole tumor cell lysate as a means topulse DCs with Ag. In fact, previous studies have shown that tumorignorance by CD8⁺ T cells can be reversed if they are exposed toantigen-pulsed DCs. (Dalyot-Herman, N., Bathe, O. F. & Malek, T. R., JImmunol 165, 6731-7, 2000) Thus, while the potential for impact onanti-tumor immunity by DCs is clear, the trafficking and survival ofthese DCs have been significant limiting factors in their use, thus anenhancement to their survival provides a critical improvement to thistherapy

Enhancing In Vivo DC Vaccines Generated from Transduced HematopoieticStem Cells

Another potential avenue for enhancing immunotherapeutic responses is byimproving DC survival in a model we have recently developed involvinggeneration of DCs in vivo from hematopoietic stem-progenitor cells(termed HSCs in this application) that have been transduced with a modeltumor Ag prior to transplantation followed by differentiation into DCsin vivo via administration of systemic agents. This method provides forefficient expression of Ag by DCs in vivo. The introduction of genesencoding Ag into the HSCs combines both effective delivery of Ag andalso the benefits of autologous BMT (autoBMT), which is an importanttreatment strategy for a number of hematologic malignancies. The successof autoBMT may be due in part to the generation of a lymphopenicenvironment in which it is easier to re-direct the immune system towardstumor antigens, as shown with vaccines administered post BMT, includingDC based vaccines. (Asavaroengchai, W., Kotera, Y. & Mule, J. J., ProcNatl Acad Sci USA 99, 931-6, 2002).

Activation of DCs is necessary to achieve a significant percentage oftumor regressors. However, activation of DCs is a double edged sword inthat while it is necessary for maximal immune stimulation, that it alsoleads to an induction of MINOR, thereby rapidly initiating cell death.Thus, inhibiting MINOR along with administration of activational agentsit is possible that we can increase the immunogenic effect of DCs. Bydetermining first whether there are differences in induction of MINOR bysome of these candidate activators, this will allow the determination ofwhich agents are most likely to produce an enhanced effect from theinhibition of MINOR.

Previous studies have shown that tumor ignorance by CD8⁺ T cells can bereversed if they are exposed to Ag-pulsed DCs (Dalyot-Herman, N., Bathe,O. F. & Malek, T. R., J Immunol 165, 6731-7, 2000), thus it is possiblethat when DCs can be kept alive long enough to activate T cells in theright setting, that their stimulatory capacity in these tolerizingsettings can be greatly enhanced. Other molecules that activate DCs,primarily immunostimulatory synthetic CpG oligonucleotides have showninteresting effects in the context of stimulating tumor immunity. TheseCpGs act through toll like receptor (TLR)9 to activate both mature andimmature DCs to upregulate co-stimulatory molecules in vitro(Sparwasser, T. et al., Eur J Immunol 28, 2045-54, 1998; Bauer, M. etal., J Immunol 166, 5000-7, 2001) and in vivo to produce γIFN (Kadowaki,N., Antonenko, S. & Liu, Y. J., J Immunol 166, 2291-5, 2001). CpG oligosalso inhibit apoptosis of DCs (Park, Y., Lee, S. W. & Sung, Y. C., JImmunol 168, 5-8, 2002), which may contribute to their observedenhancement of DC vaccines (Merad, M., Sugie, T., Engleman, E. G. &Fong, L., Blood 99, 1676-82, 2002). In addition to these DC stimulators,some more recently described agents stimulate DCs through TLR7/8(Doxsee, C. L. et al., J Immunol 171, 1156-63, 2003) and appear toproduce a potent effect, possibly working through the selectivestimulation of plasmacytoid DCs (Gibson, S. J. et al., Cell Immunol 218,74-86, 2002).

I. Definitions:

“Antigen presenting cell progenitors” refers to cells that are capableof developing into a mature antigen presenting cell, e.g., a dendriticcell. Antigen-presenting cell progenitors such as dendritic cellprogenitors include, for example, bone marrow stem cells, monocytes, andpartially differentiated cells such as CD 14+ or CD34+ cells. Antigenpresenting cell progenitors such as dendritic cell progenitors may bedifferentiated into mature cells by adding to the culture medium orstimulating the production of compounds, chemokines, and cytokines suchas GM-CSF, in addition to IL-4, TGF.beta., M-CSF, G-CSF, IL-3, IL-1,TNF.alpha., CD40 ligand, LPS, flt3 ligand, SCF, FL, protein kinase Cactivators such as phorbol ester, and CD40 ligand, etc., and/or othercompound(s), or combinations thereof, e.g., GM-CSF and IL-4; GM-CSF andTGF.beta.; GM-CSF, IL-4, and TGF.beta.; IL-3 and TNF; SCF and FL; IL-4and TNF; FL and TNF; TNF and SCF; SCF, IL-1B, IL-3, IL-4, and IL-6;TGF-.beta. and TNF; TGF-.beta. and IL-4; GM-CSF, TNF and TGF-.beta. inbovine serum free media, etc., that induce differentiation, maturation,and proliferation of antigen presenting cells (see, e.g., Paul,Fundamental Immunology (3 ed. 1993); see also Young, Curr. Opin.Hematol. 6:135-144 (1999); Agilette et al., Haematologica 83:824-848(1998); and Gluckman et al., Cytokines, Cell. and Mol. Ther. 3:187-196(1997)). “Dendritic cells” are highly potent APCs (Banchereau &Steinman, Nature 392:245-251 (1998) and have been shown to be effectiveas a physiological adjuvant for eliciting prophylacetic or therapeuticantitumor immunity (see Timmerman & Levy, Ann. Rev. Med. 50:507-529(1999)). In general, dendritic cells may be identified based on theirtypical shape (stellate in situ, with marked cytoplasmic processes(dendrites) visible in vitro), their ability to take up, process andpresent antigens with high efficiency and their ability to activate naveT cell responses. Dendritic cells may, of course, be engineered toexpress specific cell-surface receptors or ligands that are not commonlyfound on dendritic cells in vivo or ex vivo, and such modified dendriticcells are contemplated by the present invention.

Dendritic cells can be categorized as “immature” and “mature” cells,which allows a simple way to discriminate between two well-characterizedphenotypes. However, this nomenclature should not be construed toexclude all possible intermediate stages of differentiation. Immaturedendritic cells are characterized as APC with a high capacity forantigen uptake and processing, which correlates with the high expressionof Fc.gamma. receptor and mannose receptor. The mature phenotype istypically characterized by a lower expression of these markers, but ahigh expression of cell surface molecules responsible for B and T cellactivation such as class I and class II MHC molecules, adhesionmolecules (e.g., CD54, CD 18, and CD11) and costimulatory molecules(e.g., CD40, CD80, CD83, CD86 and 4-1BB).

A “cytokine or chemokine that promotes differentiation of antigenpresenting cell progenitors” refers to any cytokine or chemokine thatdrives stem cells or partially differentiated cells to a more mature ordifferentiated APC, e.g., dendritic cell, phenotype, e.g., a compoundthat drives a stem cell or a partially differentiated cell to animmature or mature dendritic cell phenotype. The cytokine can beprovided exogenously to the cell culture, or can be provided by cells inthe culture that express the cytokine or chemokine, either an endogenousor a recombinant protein. For expression of a recombinant protein, cellsin the culture are transfected with an expression vector encoding thechemokine or cytokine, which then produces the protein.

“Antigen” refers to a peptide or polypeptide comprising one or more MHCclass I or MHC class II epitopes. Thus, an antigen can be a protein orpolypeptide, fragment of a protein or polypeptide, or a peptidecomprising one or more epitopes. The antigen can be provided exogenouslyto the cell culture, or can be provided by cells in the culture theexpress the antigen, either an endogenous or recombinant protein. Forexpression of a recombinant protein, cells in the culture aretransfected with an expression vector encoding the antigen, which thenproduces the protein. The antigen may be a whole protein or fragmentthereof, or an MHC II epitope of about 8 to 25 amino acid residues, morepreferably 9-15 amino acid residues. Dendritic cells of the inventioncan be pulsed with antigen either before or after administration of theadjuvant compound of formula I.

The terms “preventing” or “inhibiting” are intended to mean a reductionin cell death or a prolongation in the survival time of the cell. Theyalso are intended to mean a diminution in the appearance or a delay inthe appearance of morphological and/or biochemical changes normallyassociated with apoptosis. Thus, this invention provides compositionsand methods to increase survival time and/or survival rate of a cell orpopulation of cells which, absent the use of the method, would normallybe expected to die. Accordingly, it also provides compositions andmethods to prevent or treat diseases or pathological conditionsassociated with unwanted cell death in a subject.

II. Cells and Cell Culture:

The present invention provides methods of culturing and inducingmaturation of antigen presenting cells (APCs) ex vivo. Specifically, thepresent invention is directed to methods for culturing and inducing thematuration of dendritic cells (DCs). In addition, the present inventionprovides methods of pulsing the cultured, matured APCs with an antigenof interest.

A. Types of Cells

Any antigen presenting cell (APC) can be used with the methods of thepresent invention. The term APC encompasses any cell capable of handlingand presenting an antigen to lymphocytes. Typically, APCs include, e.g.,Langerhans dendritic cells and Follicular dendritic cells. In addition,B cells have also been shown to have an antigen presenting function andare thus contemplated by the present invention in that should they beshown to express MINOR and that this expression led to induction ofapoptosis, that inhibition of MINOR could potentially be employed toprotect them from apoptosis. B cell based vaccinations are one possiblemeans of inducing immunity. Conversely, should the presence of B cellsbe shown to be involved in a pathologic process, augmenting MINORexpression could be viewed as one means to eliminate B cells. Inpreferred embodiments of the present invention, the APCs are dendriticcells.

B. Source of Cells

APCs can be isolated from any of the tissues where they reside and whichare known to those of skill in the art. In particular, dendritic cellsand their progenitors may be obtained from any tissue source comprisingdendritic cell precursors that are capable of proliferating and maturingin vitro into dendritic cells, when cultured and induced to matureaccording to the methods of the present invention. Such suitable tissuesources include, e.g., peripheral blood, bone marrow, tumor-infiltratingcells, peritumoral tissues-infiltrating cells, lymph node biopsies,thymus, spleen, skin, umbilical cord blood, monocytes harvested fromperipheral blood, CD34 or CD14 positive cells harvested from peripheralblood, blood marrow or any other suitable tissue or fluid. In thecontext of the present invention, dendritic cells are preferablyisolated from bone marrow or from peripheral blood mononuclear cells(PBMCs).

Peripheral blood can be collected using any standard apheresis procedureknown in the art (see, e.g., Bishop et al., Blood 83:610:616 (1994)).PBMCs can then be prepared from whole blood samples by separatingmononuclear cells from red blood cells. There are a number of methodsfor isolating PBMCs including, e.g., velocity sedimentation, isopyknicsedimentation, affinity purification, and flow cytometry. Typically,PBMCs are separated from red blood cells by density gradient (isopyknic)centrifugation, in which the cells sediment to an equilibrium positionin the solution equivalent to their own density. For density gradientcentrifugation, physiological media should be used, the density of thesolution should be high, and the media should exert little osmoticpressure. Density gradient centrifugation uses solutions such as sodiumditrizoate-polysucrose, Ficoll, dextran, and Percoll (see, e.g.,Freshney, Culture of Animal Cells, 3rd ed. (1994)). Such solutions arecommercially available, e.g., HISTOPAQUE® (Sigma). Examples of methodsfor isolating dendritic cells from PBMCs are disclosed in, e.g., U.S.Pat. Nos. 6,017,527 and 5,851,756; and in O'Doherty et al., J. Exp. Med.178:1067-1078 (1993); Young and Steinman, J. Exp. Med. 171:1315-1332(1990); Freudenthal and Steinman, Proc. Natl. Acad. Sci. USA57:7698-7702 (1990); Macatonia et al., Immunol. 67: 285-289 (1989); andMarkowicz and Engleman, J. Clin. Invest. 85:955-961 (1990).

CD34+ PBMCs or CD14+ PBMCs can further be selected as a preferred sourceof dendritic cells using a variety of selection techniques known tothose of skill in the art. For example, monoclonal antibodies (or anyprotein-specific binding protein) can be used to bind to a cell surfaceantigen found on the surface of the PBMC sub-population of interest(e.g., CD34 or CD14 on the surface of CD34+ or CD14+ PBMCs,respectively). Binding of such specific monoclonal antibodies allows theidentification and isolation of the sub-group of PBMCs of interest froma total PBMC population by any of a number of immunoaffinity methodsknown to those of skill in the art. Examples of immunoaffinity methodsfor isolating sub-populations of PBMCs are described in, e.g., U.S. Pat.No. 6,017,527.

Alternatively, the dendritic cells of the present invention can beisolated from bone marrow. For a general description of methods forisolating dendritic cells from bone marrow see, e.g., U.S. Pat. No.5,994,126; Dexter et al., in Long-Term Bone Marrow Culture, pages 57-96,Alan R. Liss, (1984); and Lutz et al., J. Immunol. Methods 223:77-92(1999). Dendritic cells from bone marrow can typically be obtained froma number of different sources, including, for example, from aspiratedmarrow. Alternatively, bone marrow can be extracted from a sacrificedanimal by dissecting out the femur, removing soft tissue from the boneand removing the bone marrow with a needle and syringe. Dendritic cellscan be identified among the different cell types present in the bonemarrow based on their morphological characteristics. For example,cultured immature dendritic cells in one or more phases of theirdevelopment are loosely adherent to plastic, flattening out with astellate shape.

In a preferred embodiment, the present invention provides methods togrow large numbers of murine dendritic cells from mouse bonemarrow-derived dendritic cell progenitors. In another preferredembodiment, the present invention provides methods to grow large numberof human dendritic cells obtained from CD14 positive human peripheralblood monocyte precursors or CD34+ progenitors.

Optionally, prior to culturing the cells, the tissue source can bepre-treated to remove cells that may compete with the proliferationand/or the survival of the dendritic cells or of their precursors.Examples of such pre-treatments are described, e.g., in U.S. Pat. No.5,994,126.

C. Number of Days

Those of skill in the art will recognize that APCs can be cultured forany suitable amount of time. Typically, APCs are cultured from 4 to 15days. In a preferred embodiment, the APCs of the invention are culturedfor 5-7 days (Inaba et al., J. Exp. Med. 176:1693 (1992); Inaba et al.,J. Exp. Med. 175:1157 (1992); Inaba et al., Current Protocols Immunol.,Unit 3.7 (Coico et al., eds. 1998); Schneider et al., J. Immunol. Meth.154:253 (1992)). In another preferred embodiment, the APCs of theinvention are cultured for 10-12 days (Lutz et al., supra).

D. Compounds Added: Adjuvants and Growth Factors

1. Cytokines and Chemokines

GM-CSF has been found to promote the proliferation in vitro of bothnonadherent immature dendritic cells and adherent macrophages (see,e.g., U.S. Pat. No. 5,994,126; and Lutz et al., supra). In the contextof the present invention, precursor dendritic cells are thus preferablycultured in the presence of GM-CSF at a concentration sufficient topromote their survival and proliferation. The dose of GM-CSF depends,e.g., on the amount of competition from other cells (especiallymacrophages and granulocytes) for the GM-CSF, and on the presence ofGM-CSF inactivators in the cell population (see, e.g., U.S. Pat. No.5,994,126). The GM-CSF concentration is typically of about 1 ng/ml to100 ng/ml, preferably of about 5 ng/ml to about 20 ng/ml. GM-CSF can beobtained from different sources well known to those of skill in the art(see, e.g., Lutz et al., supra; and U.S. Pat. No. 5,994,126).

In addition to GM-SCF, a variety of cytokines have been shown to inducethe proliferation and/or maturation of dendritic cells and other APCs,and are suitable for use with the methods of the present invention (see,e.g., Caux et al., J. Exp. Med. 180:1263-1272 (1984); Allison, ArchivumImmunologiae et Therapiae Experimentalis 45:141-147 (1997)). Cytokinesthat can be used to enhance the maturation of dendritic cells ex vivoinclude, but are not limited to, TNF-alpha, stem cell factor (SCF; alsonamed c-kit ligand, steel factor (SF), mast cell growth factor (MGF);see, e.g., EP 423,980; and U.S. Pat. No. 6,017,527), granulocytecolony-stimulating factor (G-CSF), monocyte-macrophagecolony-stimulating factor (M-CSF), as well as a number of interleukins,such as, e.g., IL-1.alpha. and IL-1.beta., IL-3, IL-4, IL-6, and IL-13(see, e.g., U.S. Pat. Nos. 6,017,527 and 5,994,126). In addition topromoting the maturation of dendritic cells, some interleukins (e.g.,IL-4) have been shown to suppress the overall growth of macrophages andthus favors higher levels of pure DC growth. Cytokines are used inamounts which are effective in increasing the proportion of dendriticcells present in the culture by enhancing either the proliferation orthe survival of dendritic cell precursors.

In preferred embodiments, the dendritic cell precursors of the presentinvention are cultured in the presence of GM-CSF. In other preferredembodiments, the dendritic cells of the present invention are culturedin the presence of both GM-CSF and IL-4. When human dendritic precursorcells are cultured, the GM-CSF is preferably human GM-CSF (huGM-CSF).

2. Adjuvants

The present invention is further based, at least in part, on thediscovery that a variety of adjuvants can be used to stimulate thematuration ex vivo of immature dendritic cells cultured as describedabove. Specifically, immature dendritic cells can be harvested from theinduction cultures described supra and their maturation to end-stageantigen presenting cells can be induced by treating the cells with avariety of adjuvants. Adjuvants that promote the maturation of dendriticcells include, but are not limited to, MPL® immunostimulant and selectedsynthetic lipid A analogs such as aminoalkyl glucosamide phosphate(AGP). Synthetic lipid A analogs include, for example, lipid Amonosaccharide synthetics such as RC-529, RC-544 and RC-527, and thedisaccharide mimetic, RC-511. These adjuvants are typically used as 10%ethanol-in-water formulations, although any other formulation thatpromotes the maturation of dendritic cells is suitable for use with themethods of the present invention. Adjuvants that can be used with themethods of the present invention can be synthesized or obtained from avariety of sources (see, e.g., Lutz et al., supra; Johnson et al.,Bioorganic Medicinal Chemistry Letters 9:2273-2278 (1999)).

E. Description of the Maturation of DCs

The maturation of DCs can be followed using a number of molecularmarkers and of cell surface phenotypic alterations. These changes can beanalyzed, for example, using flow cytometry techniques. Typically, thematuration markers are labeled using specific antibodies and DCsexpressing a marker or a set of markers of interest can be separatedfrom the total DC population using, for example, cell sorting FACSanalysis. Markers of DC maturation include genes that are expressed athigher levels in mature DCs compared to immature DCs. Such markersinclude, but are not limited to, cell surface MHC Class II antigens (inparticular HLA-DR), ICAM-1, B7-2, costimulating molecules such as CD 40,CD 80, CD 86, CD 83, cell trafficking molecules such as CD 54, CD 11cand CD 18, etc. Furthermore, mature dendritic cell can be identifiedbased on their ability to stimulate the proliferation of naiveallogeneic T cells in a mixed leukocyte reaction (MLR).

In addition, it has been shown that, in general, while immaturedendritic cells are very efficient at antigen uptake but are poorantigen presenting cells, mature dendritic cells are poor at antigenuptake but are very efficient antigen presenting cells. The antigenpresenting function of a dendritic cell can be measured usingantigen-dependent, MHC-restricted T cell activation assays as describedherein, as well as other standard assays well known to those of skill inthe art. T cell activation can further be determined, e.g., by measuringthe induction of cytokine production by the stimulated dendritic cells.The stimulation of cytokine production can be quantitated using avariety of standard techniques, such as ELISA, well known to those ofskill in the art.

F. General Cell Culture Methods

The present invention relies on routine techniques in the field of cellculture, and suitable conditions can be easily determined by those ofskill in the art (see, e.g., Freshney et al., Culture of Animal Cells,3rd ed. (1994)). In general, the cell culture environment includesconsideration of such factors as the substrate for cell growth, celldensity and cell contract, the gas phase, the medium, the temperature,and the presence of growth factors.

Exemplary cell culture conditions for dendritic cells and dendritic cellprecursors are described in, e.g., U.S. Pat. Nos. 6,017,527 and5,851,756; Inaba et al., J. Exp. Med. 176:1693 (1992); Inaba et al., J.Exp. Med. 175:1157 (1992); Inaba et al., Current Protocols Immunol.,Unit 3.7 (Coico et al., eds. 1998); Schneider et al., J. Immunol. Meth.154:253 (1992); and Lutz et al., supra.

The cells of the invention can be grown under conditions that providefor cell to cell contact. In a preferred embodiment, the cells are grownin suspension as three dimensional aggregates. Suspension cultures canbe achieved by using, e.g., a flask with a magnetic stirrer or a largesurface area paddle, or on a plate that has been coated to prevent thecells from adhering to the bottom of the dish. For example, the cellsmay be grown in Costar dishes that have been coated with a hydrogel toprevent them from adhering to the bottom of the dish.

For cells that grow in a monolayer attached to a substrate, plasticdishes, flasks, roller bottles, or microcarriers are typically used.Other artificial substrates can be used such as glass and metals. Thesubstrate is often treated by etching, or by coating with substancessuch as collagen, chondronectin, fibronectin, laminin or poly-D-lysine.The type of culture vessel depends on the culture conditions, e.g.,multi-well plates, petri dishes, tissue culture tubes, flasks, rollerbottles, microcarriers, and the like. Cells are grown at optimaldensities that are determined empirically based on the cell type.

Important constituents of the gas phase are oxygen and carbon dioxide.Typically, atmospheric oxygen tensions are used for dendritic cellcultures. Culture vessels are usually vented into the incubatoratmosphere to allow gas exchange by using gas permeable caps or bypreventing sealing of the culture vessels. Carbon dioxide plays a rolein pH stabilization, along with buffer in the cell media, and istypically present at a concentration of 1-10% in the incubator. Thepreferred CO.sub.2 concentration for dendritic cell cultures is 5%.

Cultured cells are normally grown in an incubator that provides asuitable temperature, e.g., the body temperature of the animal fromwhich is the cells were obtained, accounting for regional variations intemperature. Generally, 37.degrees.C. is the preferred temperature fordendritic cell culture. Most incubators are humidified to approximatelyatmospheric conditions.

Defined cell media are available as packaged, premixed powders orpresterilized solutions. Examples of commonly used media includeIscove's media, RPMI 1640, DMEM, and McCoy's Medium (see, e.g.,GibcoBRL/Life Technologies Catalogue and Reference Guide; SigmaCatalogue). Defined cell culture media are often supplemented with 5-20%serum, e.g., human, horse, calf, or fetal bovine serum. The culturemedium is usually buffered to maintain the cells at a pH preferably fromabout 7.2 to about 7.4. Other supplements to the media include, e.g.,antibiotics, amino acids, sugars, and growth factors (see, e.g., Lutz etal., supra).

As described above, GM-CSF is typically added in concentrations rangingfrom 5 ng/ml to about 20 ng/ml. Other factors described herein and knownto stimulate growth of dendritic cells may be included in the culturemedium. Some factors will have different effects that are dependent uponthe stage of differentiation of the cells, which can be monitored bytesting for differentiation markers specific for the cell's stage in thedifferentiation pathway. GM-CSF is preferably present in the mediumthroughout culturing. Other factors that may be desirable to add to theculture medium include, but are not limited to, granulocytecolony-stimulating factor (G-CSF), M-CSF, TNF-.alpha., IFN-.gamma.,IL-1, IL-3, IL-6, SCF, LPS, and thrombopoietin. In some embodiments ofthe present invention, IL-4 is added to the culture medium, preferablyat a concentration ranging from 1-100 ng/ml, most preferably from about5 to about 20 ng/ml.

The present invention is also based in part on the surprising resultthat dendritic cell can be recovered and used after cryogenic storage.The present invention, thus, also provides methods for cryogenicallystoring precultured DCs, e.g., in liquid nitrogen, for several weeks. Ina preferred embodiment, the dendritic cells are cultured in the presenceof GM-CSF, preferably for 10 days, prior to being stored cryogenically.The DCs can be stored either as immature cells or, preferably, asmatured APCs, following stimulation by suitable adjuvants, as describedabove. Furthermore, the DCs can be cryogenically stored either before orfollowing exposure to an antigen of interest.

A variety of cryopreservation agents can be used and are described in,e.g., U.S. Pat. No. 5,788,963. Controlling the cooling rate, addingcryoprotective agents and/or limiting the heat of fusion phase wherewater turns to ice help preserve the function of the activated DCs. Thecooling procedure can be carried out by use of, e.g., a programmablefreezing device or a methanol bath procedure. After thorough freezing,cells can be rapidly transferred to a long-term cryogenic storagevessel. The samples can be cryogenically stored, for example, in liquidnitrogen (−196.degree.C.) or its vapor (−165.degree.C.). Such storage isgreatly facilitated by the availability of highly efficient liquidnitrogen refrigerators. For a general description of methods to storeDCs cryogenically see, e.g., U.S. Pat. No. 5,788,963.

IV. Antigen Stimulation

A. Pulsing the Antigen Presenting Cells with an Antigen of Interest

Following expansion in culture and maturation, the APCs of the presentinvention can further be pulsed with an antigen. APCs pulsed with anantigen of interest will process and present epitopes of the antigen.Antigens can be from any source, including, e.g., viruses, bacteria,parasites, etc. In one embodiment, the antigen is derived fromMycobacterium sp, Chlamydia sp., Leishmania sp., Trypanosoma sp.,Plasmodium sp., or a Candida sp. APCs can be pulsed with either theentire peptide (antigen) or with a fragment thereof having immunogenicproperties, e.g., an epitope.

Briefly, the antigen-activated APCs (e.g., antigen-activated dendriticcells) of the invention are produced by exposing, in vitro, an antigento the APCs (e.g., the dendritic cells) prepared according to themethods of the invention. Dendritic cells, for example, are plated inculture dishes and exposed to an antigen of interest in a sufficientamount and for a sufficient period of time to allow the antigen to bindto the dendritic cells. The amount and time necessary to achieve bindingof the antigen to the dendritic cells may be determined by usingstandard immunoassays or binding assays. Any other method known to thoseof skill in the art may also be used to detect the presence of antigenon the dendritic cells following their exposure to the antigen. Methodsfor pulsing dendritic cells with an antigen of interest are described,e.g., in U.S. Pat. No. 6,017,527.

B. Obtaining the Antigens

In general, antigens and fragments thereof may be prepared using any ofa variety of procedures well known to those of skill in the art. Forexample, antigens can be naturally occurring and purified from a naturalsource.

Alternatively, antigens and fragments thereof can be producedrecombinantly using a DNA sequence that encodes the antigen, which hasbeen inserted into an appropriate expression vector, i.e., a vectorwhich contains the necessary elements for the transcription andtranslation of the inserted coding sequence, and expressed in anappropriate host. Methods which are well known to those skilled in theart may be used to construct expression vectors containing sequencesencoding a polypeptide of interest and appropriate transcriptional andtranslational control elements. These methods include in vitrorecombinant DNA techniques, synthetic techniques, and in vivo geneticrecombination. Such techniques are described in Sambrook et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories,Cold Spring Harbor, N.Y. (1989); and Ausubel et al., Current Protocolsin Molecular Biology (1995 supplement).

In addition, antigens and portions thereof may also be generated bysynthetic means. Synthetic polypeptides having fewer than about 100amino acids, and generally fewer than about 50 amino acids, may begenerated using techniques well known in the art. For example, suchpolypeptides may be synthesized using any of the commercially availablesolid-phase techniques, such as the Merrifield solid-phase synthesismethod, where amino acids are sequentially added to a growing amino acidchain (see Merrifield, J. Am. Chem. Soc. 85:2149-2146 (1963)). Equipmentfor automated synthesis of polypeptides is commercially available fromsuppliers such as Perkin Elmer/Applied BioSystems Division, Inc., FosterCity, Calif., and may be operated according to the manufacturer'sinstructions. Variants of a native antigen may generally be preparedusing standard mutagenesis techniques, such as oligonucleotide-directedsite-specific mutagenesis. Sections of the DNA sequence may also beremoved using standard techniques to permit preparation of truncatedpolypeptides.

Within certain embodiments, the antigen of interest may be a fusionprotein that comprises multiple polypeptides. A fusion protein may, forinstance, include an antigen and a fusion partner which may, e.g.,assist in providing T helper epitopes, and/or assist in expressing theprotein at higher yields than the native recombinant protein. Otherfusion partners may be selected so as to increase the solubility of theprotein or to enable the protein to be targeted to desired intracellularcompartments. Still further fusion partners include affinity tags, whichfacilitate the purification of the protein. Fusion proteins maygenerally be prepared using standard techniques, including chemicalconjugation. Preferably, a fusion protein is expressed as a recombinantprotein.

Furthermore, epitopes for use with the methods of the present inventioncan be selected based on the presence of specific MHC I and MHC IImotifs well known to those of skill in the art.

C. Selecting the Antigens

In the context of the present invention, the antigens, antigen fragmentsor fusion proteins used to pulse the dendritic cells are preferablyimmunogenic, i.e., they are able to elicit an immune response (e.g.,cellular or humoral) in a patient, such as a human, and/or in abiological sample (in vitro). In particular, antigens that areimmunogenic (and portions of such antigens that are immunogenic)comprise an epitope recognized by a B-cell and/or a T-cell surfaceantigen receptor. Antigens that are immunogenic (and immunogenicportions of such antigens) are capable of stimulating cellproliferation, interleukin-12 production and/or interferon-.gamma.production in biological samples comprising one or more cells selectedfrom the group of T cells, NK cells, B cells and macrophages, where thecells have been previously stimulated with the antigen.

A variety of standard assays for measuring the immunogenic properties ofa polypeptide of interest or of a portion thereof are available andknown to those of skill in the art (see, e.g., Paul, FundamentalImmunology, 3d ed., Raven Press, pp. 243-247 (1993), and referencescited therein).

V. Immune Responses Elicited by DCs

In one aspect of the invention, the activated antigen presenting cells(e.g., the activated dendritic cells) are used to generate an immuneresponse to an antigen of interest. An immune response to an antigen ofinterest can be detected by examining the presence, absence, orenhancement of specific activation of CD4+ or CD8+ T cells or byantibodies. Typically, T cells isolated from an immunized individual byroutine techniques (e.g., by Ficoll/Hypaque density gradientcentrifugation of peripheral blood lymphocytes) are incubated with anantigen. For example, T cells may be incubated in vitro for 2-9 days(typically 4 days) at 37.degree.C. with the antigen. It may be desirableto incubate another aliquot of a T cell sample in the absence of theantigen to serve as a control.

Specific activation of CD4+ or CD8+ T cells may be detected in a varietyof ways. Methods for detecting specific T cell activation includedetecting the proliferation of T cells, the production of cytokines, orthe generation of cytolytic activity (i.e., generation of cytotoxic Tcells specific for an antigen). For CD4+ T cells, a preferred method fordetecting specific T cell activation is the detection of theproliferation of T cells. For CD8+ T cells, a preferred method fordetecting specific T cell activation is the detection of the generationof cytolytic activity.

Detection of the proliferation of T cells may be accomplished by avariety of known techniques. For example, T cell proliferation can bedetected by measuring the rate of DNA synthesis. T cells which have beenstimulated to proliferate exhibit an increased rate of DNA synthesis. Atypical way to measure the rate of DNA synthesis is, for example, bypulse-labeling cultures of T cells with tritiated thymidine, anucleoside precursor which is incorporated into newly synthesized DNA.The amount of tritiated thymidine incorporated can be determined using aliquid scintillation spectrophotometer. Other ways to detect T cellproliferation include measuring increases in interleukin-2 (IL-2)production, Ca2+ flux, or dye uptake, such as3-(4,5-dimethylthiazol-2-yl-)-2,5-diphenyltetrazolium. Alternatively,synthesis of lymphokines (e.g., interferon-gamma (IFN-.gamma.)) can bemeasured or the relative number of T cells that can respond to theantigen may be quantified.

The secretion of IL-2 or IFN-.gamma. can be measured by a variety ofknown techniques, including, but not limited to, the double monoclonalantibody sandwich immunoassay technique of David et al. (U.S. Pat. No.4,376,110); monoclonal-polyclonal antibody sandwich assays (Wide et al.,in Kirkham and Hunter, eds., Radioimmunoassay Methods, E. and S.Livingstone, Edinburgh (1970)); the “western blot” method of Gordon etal. (U.S. Pat. No. 4,452,901); immunoprecipitation of labeled ligand(Brown et al., J. Biol. Chem. 255:4980-4983 (1980)); radioimmunoassays(RIA); enzyme-linked immunosorbent assays (ELISA) as described, forexample, by Raines et al., J. Biol. Chem. 257:5154-5160 (1982);immunocytochemical techniques, including the use of fluorochromes(Brooks et al., Clin. Exp. Immunol. 39:477 (1980)); and neutralizationof activity (Bowen-Pope et al., Proc. Natl. Acad. Sci. USA 81:2396-2400(1984)). In addition to the immunoassays described above, a number ofother immunoassays are available, including those described in U.S. Pat.Nos. 3,817,827; 3,850,752; 3,901,654; 3,935,074; 3,984,533; 3,996,345;4,034,074; and 4,098,876.

Methods of Preventing or Inhibiting Expression of the MINOR Gene inDendritic Cells:

A) Small Interfering RNA Technology:

Modulation of gene expression by endogenous, noncoding RNAs isincreasingly appreciated as a mechanism playing a role in eukaryoticdevelopment, maintenance of chromatin structure and genomic integrity(McManus, 2002). Recently, techniques have been developed to trigger RNAinterference (RNAi) against specific targets in mammalian cells byintroducing exogenously produced or intracellularly expressed siRNAs(Elbashir, 2001; Brummelkamp, 2002). These methods have proven to bequick, inexpensive and effective for knockdown experiments in vitro andin vivo (2 Elbashir, 2001; Brummelkamp, 2002; McCaffrey, 2002; Xia,2002). The ability to accomplish selective gene silencing now allows theuse of siRNAs to suppress gene expression for therapeutic benefit (Xia,2002; Jacque, 2002; Gitlin, 2002). In the context of the presentinvention, siRNAs have been developed which prevent or inhibit theexpression of the MINOR gene in dendritic cells, potentiating thelifespans of siRNA treated dendritic cells, and thereby increasing theirimmunogenicity.

Use of this strategy results in markedly diminished in vitro and in vivoexpression of targeted MINOR alleles. This strategy is useful inreducing expression of targeted MINOR alleles in order to modelbiological processes or to provide therapy for human diseases. Forexample, this strategy can be applied to a number of immunologicaldisorders and/or disease, including the ability to prolong the immuneresponse (i.e., inhibit MINOR expression) or diminish the immuneresponse (i.e., up-regulate MINOR expression). As used herein the term“substantial silencing” means that the mRNA of the targeted MINOR alleleis inhibited and/or degraded by the presence of the introduced siRNA,such that expression of the targeted allele is reduced by about 10% to100% as compared to the level of expression seen when the siRNA is notpresent. Generally, when an allele is substantially silenced, it willhave at least 40%, 50%, 60%, to 70%, e.g., 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, to 79%, generally at least 80%, e.g., 81%-84%, at least 85%,e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or even 100% reduction expression as compared to when the siRNA isnot present. As used herein the term “substantially normal activity”means the level of expression of an allele when an siRNA has not beenintroduced to a dendritic cell. It should be noted that others forms ofanti-sense technology are included as embodiments of the presentinvention to inhibit MINOR gene expression in dendritic cells.

B) Identification of Proteins and Small Molecules:

The present invention provides a method (also referred to herein as a“screening assay”) for identifying modulators, e.g., candidate or testcompounds or agents (e.g., peptides, peptidomimetics, small molecules,or other drugs) which have a stimulatory (increase) or inhibitory(decease) effect on the expression levels of the MINOR gene in dendriticcells.

In another embodiment, an assay is a cell-based assay in which adendritic cell which expresses the MINOR gene is contacted with a testcompound and the ability of the test compound to modulate the expressionof the MINOR gene is determined. The test compounds of the presentinvention can be obtained using any of the numerous approaches incombinatorial library methods known in the art, including: biologicallibraries; spatially addressable parallel solid phase or solution phaselibraries; synthetic library methods requiring deconvolution; theone-bead one-compound library method; and synthetic library methodsusing affinity chromatography selection. The biological library approachis limited to peptide libraries, while the other four approaches areapplicable to peptide, non-peptide oligomer or small molecule librariesof compounds (Lam, K. S. (1997) Anticancer Drug Des. 35 12:145). Aptamerlibraries may be generated as described in U.S. Pat. No. 6,423,493, U.S.Pat. No. 5,840,867, Green and Janjic, Biotechniques (2000) 39(5): 1094-6and Geyer and Brent, Methods Enzymol (2000) 328: 171-208.

Examples of methods for the synthesis of molecular libraries can befound in, for example, DeWitt et al. (1993) Proc. Natl. Acad. Sci.U.S.A. 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91:11422;Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al. (1993)Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed. Engl.33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061; andin Gallop et al. (1994) J. Med. Chem. 37:1233.

Use of these screening methods provides a means to determineagents/compounds that may alter the expression of the MINOR gene indendritic cells. These screening methods may be adapted to large-scale,automated procedures allowing for efficient high-volume screening ofpotential therapeutic agents.

VII. Pharmaceutical Compositions:

In one aspect of the invention, DCs are isolated from a patient,cultured and exposed in vitro to an antigen of interest, as describedabove, and after expansion and/or cryogenic storage are administeredback to the patient to stimulate an immune response, including T cellactivation, in vivo (see, e.g., Thurner et al., J. Immunol. Methods 223:1-15 (1999)).

The DCs obtained as described above are exposed ex vivo to an antigen,washed and administered to elicit an immune response or to augment anexisting, albeit weak, response. As such, the DCs may constitute avaccine and/or an immunotherapeutic agent. DCs presenting an antigen ofinterest can be administered using a variety of routes such as, forexample, via intravenous infusion. The immune response of the patientcan be monitored following DC administration. Infusion can be repeatedat desired intervals based upon the patient's immune response. Methodsfor administering dendritic cells to a patient for eliciting an immuneresponse in the patient are described, e.g., in U.S. Pat. Nos.5,849,589; 5,851,756; 5,994,126; and 6,017,527.

In addition, antigen presenting cells (APCs) and in particular dendriticcells can be used as delivery vehicles for administering pharmaceuticalcompositions and vaccines. In this context, the APCs may, but need not,be genetically modified, e.g., to increase the capacity for presentingthe antigen, to improve activation and/or maintenance of the T cellresponse and/or to be immunologically compatible with the receiver(i.e., matched HLA haplotype). APCs may generally be isolated from anyof a variety of biological fluids and organs as described above, and maybe autologous, allogeneic, syngeneic or xenogeneic cells.

APCs may generally be transfected with a polynucleotide encoding aantigen of interest (or portion thereof) such that the antigen, or animmunogenic portion thereof, is expressed on the cell surface. Suchtransfection may take place ex vivo, and a composition or vaccinecomprising such transfected cells may then be used for therapeuticpurposes, as described herein. Alternatively, a gene delivery vehiclethat targets a dendritic or other antigen presenting cell may beadministered to a patient, resulting in transfection that occurs invivo. In vivo and ex vivo transfection of dendritic cells, for example,may generally be performed using any methods known in the art, such asthose described in WO 97/24447, or the gene gun approach described byMahvi et al., Immunology and cell Biology 75:456-460 (1997). Antigenloading of dendritic cells may be achieved by incubating dendritic cellsor progenitor cells with the antigen, DNA (naked or within a plasmidvector) or RNA; or with antigen-expressing recombinant bacterium orviruses (e.g., vaccinia, fowlpox, adenovirus or lentivirus vectors).Prior to loading, the polypeptide may be covalently conjugated to animmunological partner that provides T cell help (e.g., a carriermolecule). Alternatively, a dendritic cell may be pulsed with anon-conjugated immunological partner, separately or in the presence ofthe polypeptide.

Vaccines and pharmaceutical compositions may be presented in unit-doseor multi-dose containers, such as sealed ampoules or vials. Suchcontainers are preferably hermetically sealed to preserve sterility ofthe formulation until use. In general, formulations may be stored assuspensions, solutions or emulsions in oily or aqueous vehicles.Alternatively, a vaccine or pharmaceutical composition may be stored ina freeze-dried condition requiring only the addition of a sterile liquidcarrier immediately prior to use.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to one of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

EXAMPLES Example 1 Analysis of Genes Upregulated in DCs

DCs have many known properties that allow them to be potent APCs. Notsurprisingly, in gene analysis, molecules such as CD86, MHCII, and CD40become upregulated upon activation. In order to elucidate other genesthat are important for DC function, a subtractive hybridization analysiswas undertaken, in which gene expression by activated DCs was comparedto that of activated macrophages.

A) MINOR Expression in Mature DCs

Initial studies utilized a subtractive hybridization strategy toidentify genes that were selectively upregulated in mature DCs relativeto activated macrophages. The goal of the subtractive hybridizationstudy was to identify new genes that were specifically upregulated inmature DCs as compared to less potent APCs (macrophages) and, thus,might contribute to the unique function of these cells.

The basic subtractive hybridization strategy for identifying genesparticular to DCs has been previously published (Tseng, S. Y. et al., JExp Med 193, 839-46, 2001). For all experiments described herein, thebasic procedure for generating DCs was as follows: bone marrow cellswere flushed from the femurs and tibias of mice, washed and cultured in100-mm dishes (1×10⁶ cells/m) in 15 ml of complete medium (RPMI-1640supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 mg ofstreptomycin, 50 mM 2-ME, and 5% FCS (all from Life Technologies)supplemented with recombinant mouse GM-CSF (1000 U/ml; R&D). Nonadherentcells were removed on days 2 and 4 and replaced with fresh medium.Depending on the experiment, either additional maturation agents orviral transduction was then conducted, as specified for each individualstudy. The initial screen identified 114 clones specifically upregulatedin DCs (data not shown), which were then further screened for redundancyand known function. Searches for homology to known genes revealed anumber of interesting candidate genes for analysis.

B) Virtual Northern Analysis of Selectively Expressed Genes

After more extensive analysis of the sequences that were upregulated inDCs, the number of candidate cDNA clones to further investigate wasnarrowed to 36. In order to confirm selective expression of genes thatwere identified by the subtractive hybridization in DCs, multipletissues from different origins were analyzed for comparative expressionof the new clones and also of some with known function (e.g., CD80 andCCR7) via virtual Northern analysis of the cDNAs from the differenttissues. MINOR expression was found to be quite selective for DCs (datanot shown).

C) Quantitative PCR Analysis of MINOR (In Vitro)

In order to confirm the relative expression levels of MINOR between DCsand activated macrophages, qPCR analysis was developed and conducted.After differentiation into either macrophages or DCs, cells were sortedfor purity, total RNA was extracted with TRIZOL, and cDNA synthesis wasconducted with reagents from Roche/Applied Biosystems, and qPCR wasconducted using the BioRad iCycler system with the standard detectionsystem and reagents for SYBR green quantification. Primers were obtainedfrom Applied Biosystems: The MINOR primer sequences are: forward:5′AGCAGCTTAAAGGACCACCA 3′ (SEQ ID NO: 4) and reverse:5′GGGTGTCAAGGAAGAGCTTG3′(SEQ ID NO: 5). Values have been normalized toactin.

D) Comparative Expression of Nurr77 and MINOR in DCs

Since MINOR is a member of the Nur77 family, efforts were made todetermine whether Nur77 was also expressed, in order to assessredundancy of these genes. qPCR analysis was conducted as above todetermine expression of Nur77 by DCs. Preparation of DCs, macrophages,and qPCR analysis was all as above. Nur77 qPCR was conducted essentiallyas described above, with primer sequences: TGATGTTCCCGCCTTTGC (SEQ IDNO. 6) and GCAAAGGCGGGAACATCA (SEQ ID NO. 7). For stimulation, LPS (25ng/ml), IL-4 (500 u/ml), or TNF-α (500 u/ml) was added to day 8 culturesfor 24-48 hrs prior to harvest of cells for RNA isolation.

As expected, expression of Nur77 was high in the thymus, consistent withits role in T cell apoptosis. However, in DCs, expression of Nur77 wasvery low to undetectable (data not shown)—even in the activated DCs, Incontrast, in unstimulated mature DCs, MINOR expression was relativelyhigh and could be induced to even higher levels by both IL-4 and TNF-α.

Example 2 MINOR Sequence Identity and Expression

MINOR is a member of the Nur77 steroid receptor family. FIG. 2 shows acartoon structure of MINOR, a 627-aa protein composed of an N-terminaltranscriptional transactivating domain, a central zinc finger DNAbinding domain with nuclear localization signals (aa290-361), andC-terminal steroid ligand binding domain (aa440-595). The figure furthershows the results of protein sequence alignment of MINOR with othermembers of the Nur77/Nurr1 steroid hormone receptor family performed, aswell as % identity to the most similar sequences.

Example 3 MINOR Expression at the Protein Level in Mature DCs

To verify that protein is translated for MINOR, various availableantibodies for this gene family were tested. An antibody that reacts tothe rat NOR-1 and mouse MINOR was identified. Utilizing this antibody,protein lysates from DCs were evaluated to assess protein expression.Mouse BMDCs were generated by standard methods and harvested at day 8.Lysates were prepared in RIPA buffer, the protein was quantified, andrun on an SDS-PAGE gel. Following transfer, the blot was probed with theanti-NOR1 antibody (Santa Cruz). FIG. 3 shows an immunoblot with thepredicted size band of 68 kDa present in mature DCs. MINOR is, indeed,expressed at the protein level in mature DCs.

Example 4 Nor-1 Expression in Mature Murine and Human DCs

BMDCs were generated from culturing bone marrow cells with GM-CSF for 6days and then cultured for another 2 days with or without any stimuli.Briefly, BM cells were flushed from the femurs and tibias of Balb/cmice, washed, and cultured in 100-mm dishes (1×10⁶ cells/ml) in 15 ml ofcomplete medium (RPMI-1640 supplemented with 2 mM L-glutamine, 100 U/mlpenicillin, 100 mg of streptomycin, Na pyruvate, Hepes, Non-essentialamino acids, 10⁻⁵ M β-ME and 5% FCS (all purchased from LifeTechnologies) supplemented with recombinant mouse GM-CSF (1000 U/ml; R&DSystems, Minneapolis, Minn.). Murine Granulocyte-macrophagecolony-stimulating factor (GM-CSF) and IL-4 were purchased from R&DSystems (Minneapolis, Minn.). All cultures were incubated at 37° C. in5% humidified CO₂. Nonadherent cells were removed on day 2 and 4 andreplaced with fresh medium. After 6 days of cultures, the cells insuspension were reseeded in complete medium with recombinant mouse IL-4(1000 U/ml; R&D) for two more days. To induce maturation of the cells,GM-CSF and IL-4 or TNF-a (50 ng/mL) were added for a further 48-hourperiod.

Mature (MHC classII highCD86high) BMDCs were FACS-sorted and examinedfor their expression of Nur77, Nor-1 and Nurr-1, compared to those ofimmature BMDCs (MHC class II low CD86 low). After differentiation intoeither macrophages or DCs, cells were sorted for purity, total RNAextracted with TRIZOL, and cDNA synthesis conducted with an Invitrogen1^(st) strand synthesis kit. 1:9 cDNA dilutions were analyzed induplicate. Standard curves were generated on the cycle threshold (Ct) vslog ng input RNA; then all samples were calculated with n=(Ct−b)/m.

For calculating fold expression of MINOR, inverse logs were calculatedand experimental values divided by 18s internal controls. AppliedBiosystems PDAR reagents were used for detection of 18s rRNA and MINORwith universal Taqman Mastermix, with primers at 600 nM and probes at200 nm. PCR was done using specific primer sets for each clone.6FAM/TAMRA labeled probe CCCTTGCAGCCCTCGCAGGTG (SEQ ID NO: 8) was usedwith flanking oligos TGCCAGCACTACGGAGTCC (SEQ ID NO: 9) andTTCTGCACCGTTCTCTTGAAGA (SEQ ID NO 10) for specific detection of MINOR.Quantitative RT-PCR analysis shows mature DCs cultured in GM-CSF orGM-CSF/IL4 expressed significantly higher levels (100 fold) of Nor-1compared to immature BMDCs (FIG. 4). Treatment of BMDCs with LPS or CpGdid not promote Nor1 expression, suggesting the protective effects ofLPS and CpG.

qPCR analysis was conducted as above to determine expression of Nur77 byDCs. Preparation of DCs, macrophages, and qPCR analysis was all asabove. For stimulation, LPS (25 ng/ml), IL-4 (500 u/ml), or TNF-α (500u/ml) was added to day 8 cultures for 24-48 hrs prior to harvest ofcells for RNA isolation.

Expression of MINOR, but not Nurr77, was detected in various DCpopulations and increased in mature DC populations. Of note, neitherNurr77 nor Nur-1 was detected in any tested DC samples.

The previous results show that Nur77 is not expressed in DCs, promptingthe hypothesis that the mouse MINOR substitutes for the signal to induceapoptosis, in a selective fashion.

Example 5 NOR-1-Induced Apoptosis in a DC-Like Cell Line

In order to determine whether forced expression of the MINOR gene wouldinduce apoptosis in a DC-like cell line, the DC 2.4 cell line, which isphenotypically similar to immature DCs (Shen, Z., Reznikoff, G.,Dranoff, G. & Rock, K. L., J Immunol 158, 2723-30, 1997) and does notexpress high levels of MINOR naturally, was gene-modified (transfected)with either a control GFP (pEGFP-N2) or the rat homolog MINOR cDNA, theNor-1 GFP vector pCI Nor-1, which was obtained from Astar Winoto andinserted into HindIII-BamHI digested peGFPN2 (Clonetech). Expression ofNor-1 in these cells was achieved by plasmid DNA transfection usingLipofectamine 2000 reagent as directed (GIBCO-BRL). 2-4 days later,cells were harvested and stained with Annexin V/7-AAD using themanufacturer's recommended protocol, and analyzed by FACS for celldeath. Such analysis included for the early (Annexin V+/AAD−)—lowerright quadrant on the FACS plot below—and late (Annexin V+/AAD+)—upperright quadrant on plot—stages of cell death. Cells were stained withPE-conjugated annexin V and 7-AAD, according to the manufacturer'sinstructions, and staining was assessed by flow cytometry. Cells thatwere apoptotic are annexin V-positive/7-AAD-negative.

Antibodies and annexin V-PE and 7-amino-actinomycin D (7-AAD) werepurchased from BD PharMingen unless specified. For the surface phenotypeanalyses described herein, the following fluorochrome-labeled mAb wereemployed: anti-CD11c (HL3), anti-Gr1 (RB6-8C5), anti-B220/CD45R(RA3-6B2), anti-CD80 (1G10), anti-CD86 (GL1), anti-1-E^(k/d) (14-4-42)mAbs (BD PharMingen, San Diego, Calif.). Prior to staining with labeledmAbs, cells were preincubated for 5 min with anti-CD 16/32 (2.4G2) mAbsto block FcγII/III receptors. Flow cytometry was performed on aFACSCalibur instrument (BD PharMingen) using CELLQUEST acquisition anddata were analyzed using the software FlowJo (Tree Star, San Carlos,Calif.).

As FIG. 5 shows, there was a significant increase in both Annexin V and7-AAD positive cells, if they constitutively expressed the Nor1 homologof MINOR, indicating that constitutive MINOR expression induces celldeath. In other words, the MINOR homolog Nor1 was shown to induceapoptosis in DC2.4 cells. It should be noted that, in all studies ofapoptosis, great care was taken in controls and compensation to minimizeartifact due to autofluorescence of dying cells.

Example 6 Generation of a Lentivirus-Based Vector Expressing siRNA toGenetically Suppress MINOR Expression

A) siRNA Design

Six different siRNAs were designed corresponding to the Nor-1 gene(GenBank accession no. NM_(—)015743). Sequences were chosen using theRNAi design software (Oligoengine, Seattle, Wash.). siRNAs with nosequence homology to any known mouse gene were used as negativecontrols. All siRNA sequences were BLAST searched in the National Centrefor Biotechnology Information's (NCBI) “search for short nearly exactmatches” mode against all mouse sequences deposited in the GenBank andwere not found to have significant homology to genes other than Nor-1.

B) Generation of Lentivirus-Based siRNA

To generate a vector-based suppression of MINOR expression, theconstruct pSUPER-retro (Oligoengine) was employed as a template. ThesiRNA oligonucleotides designed contained a sense strand of 19nucleotide sequence followed by a short spacer (TTCAAGAGA) (SEQ ID NO.7), the reverse complement of the sense strand, and five thymidines as aRNA pol III transcriptional stop signal. Briefly, the pSUPER-retrovector was digested with BglII and HindIII and the annealed oligos(5′-GATCCCCTGCCCTTGTCCGAGCTTTATTCAAGAGATAAAGCTCGGACAAGGGCATTTTTGGAAA-3′;forward (SEQ ID NO. 2) and5′-AGCTTTTCCAAAAATGCCCTTGTCCGAGCTTTATCTCTTGAATAAAGCTCGGACAAGGGCAGGG-3′;reverse (SEQ ID NO. 3)) were ligated into the vector according to themanufacturer's protocol. To construct lentivectors encoding the siRNAconstruct, the complete human H1-RNA promoter and the siRNA cassette andthe PGK promoter were subcloned at XhoI and NheI 5′ of the reporter eGFPgene of the third generation self-inactivating lentiviral vector, Sin-18provided by D. Trono (Zufferey, R. et al., J Virol 72, 9873-80, 1998).All inserts were sequenced.

A 3-plasmid transfection system was employed to generate high-titerlentivirus as previously described (Cui et al., 2003). Briefly, 293 Tcells were grown to 80% confluency on a 100-mm cell culture dish andtransfected with 10 μg of pCMV-8.9, 2.5 g of pMD.G, 5 μg of LV-siRNAusing the Lipofectamine 2000 (Life Technologies). Supernatantscontaining the virus were collected at 24 and 48 hour post-transfection,pooled and filtered with 0.2-μm filter. The titer of the virus, measuredin transducing units (TU), was determined using 293 T cells and analyzedby FACS analysis (by GFP).

C) qPCR Analysis of siRNA-MINOR-Transduced DCs

In order to validate that the siRNA was functionally decreasing theexpression of MINOR, a quantitative PCR analysis was conducted withMINOR-specific primers. DCs were generated from bone marrow progenitorcells, as described above, transduced with the LV siRNA-MINOR-GFP ondays 3-6 of culture, harvested on day 8, and evaluated by FACS forphenotype and RNA for quantitative PCR, utilizing the BioRad I cyclersystem. The MINOR primer sequences are: forward: 5′AGCAGCTTAAAGGACCACCA3′ (SEQ ID NO. 11) and reverse: 5′GGGTGTCAAGGAAGAGCTTG3′ (SEQ ID NO.12). Values were normalized to actin. FIG. 6 shows that, whileunmodified mature DCs express a high level of MINOR, transduction withsiRNA-MINOR led to a 90% knockdown in expression.

Example 7 Transduction of DC2.4 with Lentivirus ExpressingMINOR-Targeted siRNA

In order to provide further evidence that the siMINOR specificallydown-regulated MINOR expression, the ability of the siRNA-MINOR todownregulate the expression of MINOR in a controlled setting was tested.Since DC 2.4 cells normally express no detectable level of MINOR butundergo apoptosis when transduced with the rat homolog for MINOR, NOR-1,the effects of co-transducing DC2.4 cells with MINOR and the siRNA-MINORor siRNA-control were investigated.

The DC2.4 cells were transduced with siRNA-MINOR or LV-control-siRNA(MOI=5; 2 times), followed by transient transfection of rat Nor-1 intothe cells using lipofectamine 2000. Transfected cells were stained forapoptotic markers (annexin V and 7-AAD) 48 hr post-transfection.Transduced DC2.4 cells were gated based on GFP fluorescence and comparedwith the non-transduced DC2.4 (GFP negative). The expression of MINORinduced apoptosis via forced expression of MINOR, which was blocked inthe GFP+ fraction of the siRNA-MINOR transduced cells, but not in theGFP (−) fraction, or in either the GFP+ or (−) fraction of thosetransduced with MINOR and the control siRNA. NOR-1 expression wasmeasured by quantitative RT-PCR.

As shown in FIG. 7, only 10% of the DC2.4 transduced with LV-Nor1-siRNAwere dead (annexin V+, 7AAD+) whereas 66% of the cells transduced withLV-control-siRNA were annexin V+, 7AAD+. In all cases, comparable levelsof apoptosis were detected among non-transduced cells. These resultsindicate that transducing DC2.4 with lentivirus expressingMINOR-targeted siRNA prevents MINOR-induced DC apoptosis.

Example 8 Inhibition of Apoptosis in BM-Derived DCs by siRNA-MINOR

To determine whether the siRNA-MINOR would inhibit cell death in primaryBM-derived cultures, BM-DCs were generated, transduced, and followed fornatural apoptosis. Bone marrow DCs were generated in vitro, (asdescribed previously). Cells were subjected to three rounds oflentiviral transduction with concentrated virus (i.e. MOI=5) on day 2, 4and 6 in the presence of 8 μg/ml polybrene.

After 6 days of cultures, the cells in suspension were reseeded incomplete medium with and on days 5 & 6 were transduced with the LVencoding the siRNA-MINOR-GFP or control siRNA-GFP (sequenceGTATACGTGTTTGCTCCCTT (SEQ ID NO. 13), no known homology to any gene). Onday 9, the cells were analyzed for induction of cell death. As the plotsdepicted in FIG. 8 show, there is no significant difference in celldeath in the GFP⁻ fractions in both populations or the GFP+ in thecontrol. In contrast, there is a significant decrease in cell death inthe transduced (GFP⁺) fraction in the siRNA-MINOR-expressing population,indicating an inhibition of apoptosis. Accordingly, it is indicated thattransduction with the siRNA can prolong DC survival.

The previous results showing that MINOR's forced expression inducesapoptosis, and that its inhibition inhibits natural apoptosis in DCs,indicate that MINOR merits further investigation as a potentiallysignificant gene both for basic DC function, in that it may helpregulate DC lifespan, thereby limiting uncontrolled T cell activation,and also as a target for improving DC-based therapies.

Example 9 Transduction of Ex Vivo-Generated DCs with siRNA-MINOR andtheir Survival after Infusion

To test the hypothesis that the observed decrease in apoptosiscorrelates with an improved survival in vivo, BALB/c BMDCs were grown exvivo, transduced with either the GFP control or the siMINOR-GFP on days4 and 5, were sorted for GFP⁺/CD11c⁺, and then 5×10⁶ were infused on day7. Four days after infusion, the mice (3/grp) were sacrificed, and thelymph nodes were harvested and analyzed for retention of labeled infusedDCs to determine if there was a difference between the control andsiMINOR-transduced DCs in their maintenance after infusion. Lymph nodeswere enriched for DCs and then stained for CD11c. FIG. 9 shows anapproximately 3-fold increase in the survival of infused DCs thatexpressed the siRNA for MINOR. This indicates that transduction of exvivo-generated DCs with siRNA-MINOR improves their survival afterinfusion.

The use of ex vivo DC vaccines has the potential advantage of Ag loadingwith multiple Ags, some of which are not identified, through the use ofwhole tumor cell lysate as a means to pulse DCs with Ag. In fact,previous studies have shown that tumor ignorance by CD8⁺ T cells can bereversed if they are exposed to antigen-pulsed DCs. (Dalyot-Herman, N.,Bathe, O. F. & Malek, T. R., J Immunol 165, 6731-7, 2000). Thus, whilethe potential for impact on anti-tumor immunity by DCs is clear, thetrafficking and survival of these DCs have been significant limitingfactors in their use. Accordingly, an enhancement to their survivalcould provide a critical improvement to this therapy. Improving thesurvival of the DC vaccines while they are being expanded in vitro and,also, after they have been infused, is further contemplated herein.

Example 10 Transduction of Ex Vivo DCs with siRNA-MINOR and theirVaccine Potency

In order to follow the immune responses generated by the DCs, thewell-defined Influenza hemagglutinin antigen (HA) may be used, alongwith well-characterized anti-HA TCR transgenic (Tg) mice, to explorebasic immunological processes relevant to tumor immunity. The HA Ag andthe pair of MHC class I (K^(d))-(termed clone 4) and II(1-E^(d))-restricted (termed 6.5), HA-specific TCR Tg mice are utilizedto track immune responses.

Of significance to the tumor studies proposed, HA behaves like a naturaltumor Ag in a number of tumor models such as the A20 B cell lymphoma, inthat moderate levels of HA expression do not alter the biology,immunogenicity, or in vivo growth characteristics of the tumor(Sotomayor, E. M., Borrello, I., Tubb, E., Allison, J. P. & Levitsky, H.I., Proc Natl Acad Sci USA 96, 11476-81, 1999; Staveley-O'Carroll, K. etal., Proc Natl Acad Sci USA 95, 1178-83, 1998; Adler, A. J. et al., JExp Med 187, 1555-64, 1998). In addition, transgenic mice available thatexpress HA as self allow investigation of the activation of tolerant Tcells (Adler, A. J. et al., J Exp Med 187, 1555-64, 1998). Thus, whilethis Ag may have limitations, it provides a strong foundation on whichto develop this system and addresses some important fundamentalquestions of immune responses.

Since it appeared that transduction with siRNA-MINOR could inhibit celldeath, it was hypothesized that it would also enhance the immunogenicityof BM-DCs when used as a vaccine. To assess the immunogenicity of exvivo-generated, HA-pulsed DCs, DCs were generated from BM, transducedwith either GFP or siRNA-MINOR GFP, and pulsed with the class IIrestricted peptide for HA. To track the T cell responses, 6.5 T cellswere adoptively transferred (here on a thy1.1 background), and, 5 dayslater, the mice were sacrificed to determine T cell expansion in spleenand lymph nodes.

FIG. 10 shows a representative FACS plot, stained for CD4 and thy1.1 forthe siRNA-MINOR (left) and control siRNA-GFP (right), as well as a bargraph showing the average and SD for all mice (2 expts, 6 mice/vector).The lower graph shows that there was a significant enhancement instimulation of Ag-specific T cells, if the DCs expressed siRNA-MINORalong with Ag (HA in this case). Thus, siRNA-MINOR transduction of exvivo-generated, HA-pulsed DCs enhances their immunogenicity.

In these first studies, DCs were not sorted prior to infusion; thus,only a fraction (20-30%) of the infused DCs actually expressed the gene.Additional experiments contemplated herein will determine the effect forsorted DCs. Furthermore, in order to expand the system to investigatetherapeutic avenues, an A20 lymphoma has been developed that expressesthe Epstein Barr Virus Ag, LMP2. Thus, a second, naturally occurring, Agin a tumor is contemplated for future studies.

Example 11 Transduction of Ex Vivo-Generated DCs with siRNA-MINOR andtheir Capacity to Stimulate Tolerant T Cells

It was next tested whether the observed enhancement in T cellstimulation gained from transduction of ex vivo-generated DCs withsi-MINOR would also result in a stronger stimulation, perhaps sufficientto activate tolerant T cells. For these studies, a similar experimentaldesign was conducted as above, with the major difference being that theT cells used for activation were from mice that express HA as selfantigen, and followed by a thy 1.1/1.2 disparity rather than by atransgenic T cell receptor antibody. Thus the stimulation of theendogenous, tolerant repertoire is measured, rather than stimulation ofthe transgenic T cell receptor.

2.5×10⁷ T cells from C3-HA (thy 1.2) mice were adoptively transferredinto the parent strain, B10.D2 mice that carry the thy1.1 marker. B10.D2(thy1.1) DCs were grown ex vivo from bone marrow, as before, andtransduced with either the siRNA-MINOR or the siRNA-control, and then,prior to subcutaneous injection, pulsed with HA class II peptide andwashed. 5 days later, mice were sacrificed and lymph nodes removed foranalysis. Cells were stained with antibodies for thy 1.2 and CD4. Todetermine whether, indeed, these are antigen-specific responses withoutusing transgenic T cells, the control of unpulsed DCs was added.

Results of these studies (FIG. 11) show that, while ex vivo unpulsedsiRNA-MINOR-transduced DCs, or pulsed DCs that are control-transduced,are not sufficient to overcome the unresponsiveness in these mice, exvivo pulsed DCs expressing siRNA-MINOR do have sufficient stimulatorycapacity to overcome this unresponsiveness. The numbers of T cells ofexpansion in the control group is that of background detection, as showncompared to unpulsed stimulation levels, indicating that siRNA-MINORtransduction helps to impart a stronger stimulus to the DCs. In thesestudies, the C3-HA mice (termed 142) that express HA as a self-antigenwere used as the source of adoptively transferred T cells. Theperipheral T cells from the C3-HA mice are tolerant to HA byunresponsiveness, rather than deletion (Adler, A. J. et al., J Exp Med187, 1555-64, 1998). Thus, transduction of ex vivo-generated DCs withsiRNA-MINOR was found to enhance their capacity to stimulate tolerant Tcells.

Example 12 Activation of DCs Leads to Upregulation of MINOR In Vivo

In light of the upregulation of MINOR after stimulation shown in vitro,it was next tested whether there was likewise an upregulation in vivoafter systemic DC activation. BALB.c mice were treated with the TLR 7/8agonist 3M-019 (obtained from 3M Pharmaceuticals and injected at 200μg/mouse/day on days 0 and 2). Four days later, lymph nodes wereharvested from both naïve mice and the stimulated mice and sorted intothe following fractions: for the naïve mice, the LN were sorted intoCD11c^(low)/CD86^(low) or CD11c^(high)/86^(low) fractions in order toisolate the less mature naïve DCs, which were then re-checked by FACSfor purity, followed by RT-qPCR analysis. Similarly, the LN from theTLR-activated mice were sorted into CD11c^(low)/CD86^(high) orCD11c^(high)/86^(high) fractions and subjected to the above process, aswell.

Thus, the fractions were sorted based on the intensity of their CD11c(which correlates with the plasmacytoid (low) and myeloid (CD11c high)phenotypes). FIG. 12 shows that the activation of DCs leads toupregulation of MINOR in vivo. In fact, both fractions from theTLR-activated mice contain the highest levels of MINOR. However,additional studies will more accurately separate the phenotypes todetermine differences in MINOR expression.

Example 13 BMT with siRNA-MINOR-Transduced HSCs Leads to StableKnockdown of MINOR in DCs In Vivo after Reconstitution

Previously presented results using the BMT system depend on being ableto correlate GFP expression with decreased MINOR expression. It was,thus, sought to confirm that in the DC populations that were GFP+ aftertransplant with siRNA-MINOR, there was, in fact, a decrease in MINORexpression at the timepoints selected for analysis. A potential avenuefor enhancing immunotherapeutic responses is by improving DC survival ina model recently developed involving generation of DCs in vivo fromhematopoietic stem-progenitor cells (termed HSCs in this application)that have been transduced with a model tumor Ag prior to transplantationfollowed by differentiation into DCs in vivo via administration ofsystemic agents. This method provides for efficient expression of Ag byDCs in vivo.

The introduction of genes encoding Ag into the HSCs combines botheffective delivery of Ag and also the benefits of autologous BMT(autoBMT), which is an important treatment strategy for a number ofhematologic malignancies. The success of autoBMT may be due in part tothe generation of a lymphopenic environment in which it is easier tore-direct the immune system towards tumor antigens, as shown withvaccines administered post BMT, including DC based vaccines(Asavaroengchai, W., Kotera, Y. & Mule, J. J., Proc Natl Acad Sci USA99, 931-6, 2002). For all experiments describing the in vivo generationof DCs from transduced HSCs used for BMT, the following experimentalprocedure was conducted: BALB/c BM was harvested and enriched for HSCsusing the StemSep separation kit (Stem Cell Technologies). HSCs weretransduced for 3 days with either the control siRNA-GFP LV or thesiRNA-MINOR GFP and then transplanted into myeloablatively (850 cGyradiated) treated BALB/c mice. After a minimum of 8-10 weeks ofengraftment, the mice were sacrificed and their spleens and lymph nodesharvested for DC isolation.

Mice (6/Group) were transplanted with HSCs transduced with eithersiMINOR (1203) or the control GFP vector, allowed to engraft for 10weeks, then analyzed for knockdown. Spleen and lymph nodes wereharvested from the mice, stained with CD11c and B220, then FACS sortedinto GFP+ cells (thus only transduced in each group are compared here)in the plasmacytoid phenotype (CD11c^(lo)/B220⁺) or myeloid(CD11^(hi)/B220⁻). Purity of the sort was confirmed by FACS, then RNAwas isolated, cDNA transcribed, and qPCR conducted. Values werenormalized to actin, and the lowest value was arbitrarily set to 1. FIG.13 shows a significant decrease in expression of MINOR RNA in the GFP⁺population of those expressing the siRNA-MINOR vector, compared to thecontrol.

Example 14 Expression of siRNA-MINOR in CD11c+ Cells

In order to assess relative expression of the siRNA-MINOR in CD11c+cells, compared to control siRNA expression, FACS analysis was used tocompare GFP by CD11c. DC subpopulations were prepared from spleens andlymph nodes of BALB/c mice using methods similar to those describedpreviously (Sparwasser, T. et al., Eur J Immunol 28, 2045-54, 1998;Bauer, M. et al., J Immunol 166, 5000-7, 2001). Briefly, spleens werecut into small fragments, subjected to digestion by collagenase andDNase I (Roche Applied Science, Indianapolis, Ind.) at room temperaturefor 25 min, then treated with EDTA for 5 min. Light density cells wereisolated by centrifugation in Nycodenz medium (Accurate Chemical andScientific Corporation, Westbury, N.Y.), followed by depletion of CD3⁺and CD19⁺ cells, then immunomagnetic bead enrichment of CD11c⁺ cells(CD11c⁺ isolation kit, Miltenyi Biotec, Auburn, Calif.). Collagenasedigestion was omitted to isolate DCs from lymph nodes. The cells werethen stained for CD11c and 7-AAD.

FIG. 14 shows, on the right, representative FACS plots of LN for GFP byCD11c, in order to compare relative expression of the vectors in DC(upper 2 quads) vs. non-DC (lower 2 quads) populations. The statisticsshow that, while there is no significant difference in the GFPexpression by CD11c+ and CD11c− cells in the control group (labeled1203c on the FACS), there is a significant difference in the siRNA-MINORgroup (labeled 1203).

If no selective expression were present, then the ratio of GFP in CD11c+ to CD11c− should be equal. However, a comparison of the percentageof CD11c+ cells that contained the vector with the percentage of CD11c−cells that contained vector showed that the there was preferentialvector expression of siRNA-MINOR in CD 11c+/CD11c− compared to controlGFP+ in CD11c+/CD11c−. In other words, there was a selective expressionof siRNA-MINOR in CD11c+ cells, consistent with the notion thatsiRNA-MINOR confers a survival advantage to DCs, thereby preserving theCD11c population. Thus, transduction of HSC with MINOR siRNA confersselective protection to DC populations.

Example 15 siRNA-MINOR Expression in CD86^(hi) Cells

In order to assess levels of expression as related to DC maturity, asimilar analysis was conducted for expression of the vectors based onCD86 expression. Again, LN cells were enriched for DCs, and then stainedwith CD86 and 7-AAD. The ratios of GFP+ cells in the CD86^(hi) andCD86^(lo-intmdt) were compared. The ratios of upper left to lower leftshown in FIG. 15 should be the same as upper right to lower right, if noselective expression were involved. As the comparisons show, however,while in the GFP control, there is no significant difference in theseratios, in the siRNA-MINOR, there is a much higher percentage of GFP+cells in the CD86^(hi) group, indicating that this group is the mostselectively affected. This indicates that siRNA-MINOR is preferentiallymaintained in CD86^(hi) populations.

Example 16 siRNA-MINOR Increases the Viability of DC Progeny ofTransduced HSCs

Following reconstitution, mice were analyzed for the percentage ofCD11c⁺ cells that were alive. Two separate comparisons were made: Onewithin each group of mice (control GFP⁺ vs control GFP⁻ and siRNA-MINORGFP⁺ vs siRNA-MINOR GFP⁺ As FIG. 16 shows, there is a significantdifference between the MINOR-siRNA GFP⁺ and GFP⁻ and the GFP⁺ vs controlGFP⁺, indicating a selective protection from the siRNA-MINOR. Thus,siRNA transduction of HSCs prior to transplant results in a decrease inDC death.

Example 17 Human DC MINOR Expression Patterns

To address the relevance of these findings to the clinical setting,namely, inhibiting MINOR in ex vivo DC vaccines, human DCs weregenerated to determine whether the same pattern of specific expressionof MINOR was present in human cells. In effect, the studies describedfor mouse were repeated with human cells. In order to maximizesensitivity in the qPCR analysis, new primers were designed for human

MINOR with the following sequence: forward: 5′ GTA TCC AGA AGCTGG GCA GA(SEQ ID NO. 13) and reverse: 5′ CTG AAG TCG ATG CAG GAC AA (SEQ ID NO.14). Expression (normalized to actin) was compared in the following celltypes: CD34⁺ hematopoietic cell progenitors, LPS activated-monocytederived-macrophages, and activated dendritic cells.

The CD34⁺ cells were obtained at 90% purity; the monocytes and dendriticcells were generated from PBMCs, grown in the presence of M-CSF (formonocytes) or GM-CSF (30 ng/ml)+IL-4 (30 U/ml) (for DCs). At day 7, 100ng/ml of LPS was added to the cultures for activation, and cells wereharvested on day 8 for FACS analysis (to assure CD14−/CD11c+ phenotype)and RNA isolation. FACS analysis confirmed >90% purity of the two cellpopulations. CD34 cells and macrophages were tested for comparison. RNAwas transcribed into cDNA, which was used for quantitative PCR analysis.FIG. 17 shows that there is a dramatic upregulation of MINOR inactivated DCs, as previously found in the murine system.

REFERENCES

-   1. Tseng, S. Y. et al. B7-DC, a new dendritic cell molecule with    potent costimulatory properties for T cells. J Exp Med 193, 839-46    (2001).-   2. Zufferey, R. et al. Self-inactivating lentivirus vector for safe    and efficient in vivo gene delivery. J Virol 72, 9873-80 (1998).-   3. Borges, L. et al. Synergistic action of fms-like tyrosine kinase    3 ligand and CD40 ligand in the induction of dendritic cells and    generation of antitumor immunity in vivo. J Immunol 163, 1289-97    (1999).-   4. Marshall, J. L. et al. Phase I study in advanced cancer patients    of a diversified prime-and-boost vaccination protocol using    recombinant vaccinia virus and recombinant nonreplicating avipox    virus to elicit anti-carcinoembryonic antigen immune responses. J    Clin Oncol 18, 3964-73 (2000).-   5. Morse, M. A. et al. Immunotherapy with autologous, human    dendritic cells transfected with carcinoembryonic antigen mRNA.    Cancer Invest 21, 341-9 (2003).-   6. Ridgway, D. The first 1000 dendritic cell vaccines. Cancer Invest    21, 873-86 (2003).-   7. Leverkus, M. et al. Maturation of dendritic cells leads to    up-regulation of cellular FLICE-inhibitory protein and concomitant    down-regulation of death ligand-mediated apoptosis. Blood 96,    2628-31 (2000).-   8. McLellan, A. et al. MHC class II and CD40 play opposing roles in    dendritic cell survival. Eur J Immunol 30, 2612-9 (2000).-   9. Wong, B. R. et al. TRANCE (tumor necrosis factor [TNF]-related    activation-induced cytokine), a new TNF family member predominantly    expressed in T cells, is a dendritic cell-specific survival factor.    J Exp Med 186, 2075-80 (1997).-   10. Eggert, A. A. et al. Biodistribution and vaccine efficiency of    murine dendritic cells are dependent on the route of administration.    Cancer Res 59, 3340-5 (1999).-   11. Cayeux, S. et al. Direct and indirect T cell priming by    dendritic cell vaccines. Eur J Immunol 29, 225-34 (1999).-   12. Hayakawa, Y. et al. NK cell TRAIL eliminates immature dendritic    cells in vivo and limits dendritic cell vaccination efficacy. J    Immunol 172, 123-9 (2004).-   13. Kamath, A. T., Henri, S., Battye, F., Tough, D. F. &    Shortman, K. Developmental kinetics and lifespan of dendritic cells    in mouse lymphoid organs. Blood 100, 1734-41 (2002).-   14. Camporeale, A. et al. Critical impact of the kinetics of    dendritic cells activation on the in vivo induction of    tumor-specific T lymphocytes. Cancer Res 63, 3688-94 (2003).-   15. Gorski, K. et al. A set of genes selectively expressed in murine    dendritic cells: utility of related cis-acting sequences for    lentiviral gene transfer. Mol Immunol In press (2003).-   16. Cheng, L. E., Chan, F. K., Cado, D. & Winoto, A. Functional    redundancy of the Nur77 and Nor-1 orphan steroid receptors in T-cell    apoptosis. Embo J 16, 1865-75 (1997).-   17. Liu, Z. G., Smith, S. W., McLaughlin, K. A., Schwartz, L. M. &    Osborne, B. A. Apoptotic signals delivered through the T-cell    receptor of a T-cell hybrid require the immediate-early gene nur77.    Nature 367, 281-4 (1994).-   18. Kim, S. O., Ono, K., Tobias, P. S. & Han, J. Orphan nuclear    receptor Nur77 is involved in caspase-independent macrophage cell    death. J Exp Med 197, 1441-52 (2003).-   19. Kuang, A. A., Cado, D. & Winoto, A. Nur77 transcription activity    correlates with its apoptotic function in vivo. Eur J Immunol 29,    3722-8 (1999).-   20. Brenner, C. & Kroemer, G. Apoptosis. Mitochondria—the death    signal integrators. Science 289, 1150-1 (2000).-   21. Li, H. et al. Cytochrome c release and apoptosis induced by    mitochondrial targeting of nuclear orphan receptor TR3. Science 289,    1159-64 (2000).-   22. Nopora, A. & Brocker, T. Bcl-2 controls dendritic cell longevity    in vivo. J Immunol 169, 3006-14 (2002).-   23. Pirtskhalaishvili, G. et al. Transduction of dendritic cells    with Bcl-xL increases their resistance to prostate cancer-induced    apoptosis and antitumor effect in mice. J Immunol 165, 1956-64    (2000).-   24. Sotomayor, E. M., Borrello, I., Tubb, E., Allison, J. P. &    Levitsky, H. I. In vivo blockade of CTLA-4 enhances the priming of    responsive T cells but fails to prevent the induction of tumor    antigen-specific tolerance. Proc Natl Acad Sci USA 96, 11476-81    (1999).-   25. Staveley-O'Carroll, K. et al. Induction of antigen-specific T    cell energy: An early event in the course of tumor progression. Proc    Natl Acad Sci USA 95, 1178-83 (1998).-   26. Adler, A. J. et al. CD4+ T cell tolerance to parenchymal    self-antigens requires presentation by bone marrow-derived    antigen-presenting cells. J Exp Med 187, 1555-64 (1998).-   27. Dalyot-Herman, N., Bathe, O. F. & Malek, T. R. Reversal of CD8+    T cell ignorance and induction of anti-tumor immunity by    peptide-pulsed APC. J Immunol 165, 6731-7 (2000).-   28. Strome, S. E. et al. Strategies for antigen loading of dendritic    cells to enhance the antitumor immune response. Cancer Res 62,    1884-9 (2002).-   29. Schnurr, M. et al. Apoptotic Pancreatic Tumor Cells Are Superior    to Cell Lysates in Promoting Cross-Priming of Cytotoxic T Cells and    Activate NK and gammadelta T Cells. Cancer Res 62, 2347-52 (2002).-   30. Lambert, L. A., Gibson, G. R., Maloney, M. & Barth, R. J., Jr.    Equipotent generation of protective antitumor immunity by various    methods of dendritic cell loading with whole cell tumor antigens. J    Immunother 24, 232-6 (2001).-   31. Asavaroengchai, W., Kotera, Y. & Mule, J. J. Tumor lysate-pulsed    dendritic cells can elicit an effective antitumor immune response    during early lymphoid recovery. Proc Natl Acad Sci USA 99, 931-6    (2002).-   32. Parajuli, P. et al. Flt3 ligand and granulocyte-macrophage    colony-stimulating factor preferentially expand and stimulate    different dendritic and T-cell subsets. Exp Hematol 29, 1185-93    (2001).-   33. Fong, L. et al. Altered peptide ligand vaccination with Flt3    ligand expanded dendritic cells for tumor immunotherapy. Proc Natl    Acad Sci USA 98, 8809-14 (2001).-   34. Gilliet, M. et al. The development of murine plasmacytoid    dendritic cell precursors is differentially regulated by FLT3-ligand    and granulocyte/macrophage colony-stimulating factor. J Exp Med 195,    953-8 (2002).-   35. Diehl, L. et al. CD40 activation in vivo overcomes    peptide-induced peripheral cytotoxic T-lymphocyte tolerance and    augments anti-tumor vaccine efficacy. Nat Med 5, 774-9 (1999).-   36. Sotomayor, E. M. et al. Conversion of tumor-specific CD4+ T-cell    tolerance to T-cell priming through in vivo ligation of CD40. Nat    Med 5, 780-7 (1999).-   37. Cui, Y. et al. Immunotherapy of established tumors using bone    marrow transplantation with antigen gene-modified hematopoietic stem    cells. Nat Med 9, 952-958 (2003).-   38. Guerder, S. & Matzinger, P. A fail-safe mechanism for    maintaining self-tolerance. J Exp Med 176, 553-64 (1992).-   39. Sparwasser, T. et al. Bacterial DNA and immunostimulatory CpG    oligonucleotides trigger maturation and activation of murine    dendritic cells. Eur J Immunol 28, 2045-54 (1998).-   40. Bauer, M. et al. Bacterial CpG-DNA triggers activation and    maturation of human CD11c−, CD123+ dendritic cells. J Immunol 166,    5000-7 (2001).-   41. Kadowaki, N., Antonenko, S. & Liu, Y. J. Distinct CpG DNA and    polyinosinic-polycytidylic acid double-stranded RNA, respectively,    stimulate CD11c− type 2 dendritic cell precursors and CD11c+    dendritic cells to produce type I IFN. J Immunol 166, 2291-5 (2001).-   42. Park, Y., Lee, S. W. & Sung, Y. C. Cutting Edge: CpG DNA    inhibits dendritic cell apoptosis by up-regulating cellular    inhibitor of apoptosis proteins through the    phosphatidylinositide-3′-OH kinase pathway. J Immunol 168, 5-8    (2002).-   43. Merad, M., Sugie, T., Engleman, E. G. & Fong, L. In vivo    manipulation of dendritic cells to induce therapeutic immunity.    Blood 99, 1676-82 (2002).-   44. Doxsee, C. L. et al. The immune response modifier and Toll-like    receptor 7 agonist S-27609 selectively induces IL-12 and TNF-alpha    production in CD11c+CD11b+CD8− dendritic cells. J Immunol 171,    1156-63 (2003).-   45. Gibson, S. J. et al. Plasmacytoid dendritic cells produce    cytokines and mature in response to the TLR7 agonists, imiquimod and    resiquimod. Cell Immunol 218, 74-86 (2002).-   46. Nakano, H., Yanagita, M. & Gunn, M. D. CD11c(+)B220(+)Gr-1(+)    cells in mouse lymph nodes and spleen display characteristics of    plasmacytoid dendritic cells. J Exp Med 194, 1171-8 (2001).-   47. Shen, Z., Reznikoff, G., Dranoff, G. & Rock, K. L. Cloned    dendritic cells can present exogenous antigens on both MHC class I    and class II molecules. J Immunol 158, 2723-30 (1997).-   48. Langenkamp, A., Messi, M., Lanzavecchia, A. & Sallusto, F.    Kinetics of dendritic cell activation: impact on priming of TH1, TH2    and nonpolarized T cells. Nat Immunol 1, 311-6 (2000).-   49. Matzinger, P. The JAM test. A simple assay for DNA fragmentation    and cell death. J Immunol Methods 145, 185-92 (1991).-   50. Borrello, I. et al. Sustaining the graft-versus-tumor effect    through posttransplant immunization with granulocyte-macrophage    colony-stimulating factor (GM-CSF)producing tumor vaccines. Blood    95, 3011-9 (2000).

1. A method for inhibiting apoptosis in dendritic cells comprising theadministration to the dendritic cells an agent which prevents orinhibits the expression of the MINOR gene in said dendritic cells.
 2. Amethod of claim 1 wherein said agent is administered to the dendriticcells ex vivo.
 3. A method of claim 1 wherein said agent is administeredto the dendritic cells in vivo.
 4. A method of claim 1 wherein saidagent is a small interfering RNA.
 5. A method of claim 1 wherein saidagent is an anti-sense nucleotide molecule.
 6. A method of claim 4wherein the small interfering RNA is comprised of the double strandednucleotide sequence of 5′GATCCCCTGCCCTTGTCCGAGCTrTATTCAAGAGATAAAGCTCGGACAAGGGCATTTTTGGAAA-3′ (SEQID NO: 2); forward and5′AGCTTTTCCAAAAATGCCCTTGTCCGAGCTTTATCTCTTGAATAAAGCTCGGACAAGGGCAGGG-3′(SEQID NO: 3); reverse.
 7. A method of claim 1 wherein said agent inhibitssignal transduction leading to the expression of MINOR.
 8. A method ofclaim 1 wherein said agent inhibits the intracellular activity of MINOR.9. The method of claim 1, wherein the expression of MINOR in thedendritic cells is decreased by transduction of the cells with alentiviral vector encoding an siRNA construct having substantialsequence homology to MINOR.
 10. A method for decreasing the expressionof a protein in a cell population, said method comprising the steps ofgenerating a lentiviral vector encoding an siRNA construct havingsubstantial sequence homology to said protein, and transducing the cellpopulation with said lentiviral vector.
 11. The method of claim 10,wherein the protein is MINOR.
 12. The method of claim 1, wherein thedendritic cells are bone marrow dendritic cells.
 13. A method forincreasing the survival time of ex vivo-generated dendritic cellsfollowing infusion of said cells into a subject, the method comprisingthe ex vivo transduction of said cells with a lentiviral vector encodingan siRNA construct having substantial sequence homology to MINOR andinfusing the transduced cells into the subject.
 14. The method of claim13, wherein the subject is human.
 15. The method of claim 13, whereinthe dendritic cells are bone marrow dendritic cells.
 16. A method forenhancing the antigen presenting ability of dendritic cells, said methodcomprising transducing said cells with a lentiviral vector encoding ansiRNA construct having substantial sequence homology to MINOR.
 17. Themethod of claim 16, wherein the dendritic cells are bone marrowdendritic cells.
 18. A method for enhancing the capacity for dendriticcells to stimulate tolerant T cells, said method comprising transducingsaid cells with a lentiviral vector encoding an siRNA construct havingsubstantial sequence homology to MINOR.
 19. The method of claim 18,wherein the dendritic cells are bone marrow dendritic cells.
 20. Adendritic cell-based vaccine comprising dendritic cells expressingsiRNA's having substantial sequence homology to MINOR.
 21. The vaccineof claim 20, wherein said vaccine is for cancer, viral disease,bacterial disease, or immune disorders.
 22. The vaccine of claim 21,wherein said vaccine is for cancer.
 23. A method for preparing thevaccine of claim 20, comprising the step of preparing an siRNA constructhaving substantial sequence homology to MINOR and ex vivo transducingdendritic cells with said construct.
 24. A method of preserving theCD11c+ population of dendritic cells, comprising transducinghematopoietic stem-progenitor cells with a lentiviral vector encoding ansiRNA construct having substantial sequence homology to MINOR.
 25. Amethod for stably decreasing or substantially suppressing the expressionof MINOR in dendritic cells, said method comprising the steps oftransducing hematopoietic stem-progenitor cells with a lentiviral vectorencoding an siRNA construct having substantial sequence homology toMINOR and transplanting the transduced cells into a myeloablativelytreated mammalian subject.
 26. The method of claim 20, wherein themammalian subject is human.
 27. A method of augmenting an immuneresponse specific for an antigen in an individual, comprising the stepsof: (a) obtaining dendritic cells from the individual; (b) causing thedendritic cells to express the antigen by either (i) exposing thedendritic cells to the antigen in culture under conditions promotinguptake and processing of the antigen, or (ii) transfecting the dendriticcells with a gene encoding the antigen; (c) activating theantigen-expressing dendritic cells, (d) treating the dendritic cellswith an agent that inhibits MINOR expression; and. (e) administering theactivated, antigen-expressing dendritic cells to the individual.
 28. Themethod of claim 27, wherein the dendritic cells are obtained byobtaining hematopoietic stem or progenitor cells from the individual,and contacting the hematopoietic stem or progenitor cells with an agentselected from the group consisting of flt-3 ligand, GM-CSF, IL-4,TNF-.alpha., IL-3, c-kit ligand, fusions of GM-CSF and IL-3, andcombinations thereof.
 29. The method of claim 27 wherein the agentinhibiting MINOR expression is a small interfering RNA.
 30. The methodof claim 27 wherein the agent inhibits signal transduction in dendriticcells resulting in the expression of MINOR.
 31. The method of claim 27wherein the agent inhibits the intracellular activity of MINOR.
 32. Asmall interfering RNA comprising the double stranded nucleotide sequenceof5′GATCCCCTGCCCTTGTCCGAGCTTTATTCAAGAGATAAAGCTCGGACAAGGGCATTTTTGGAAA-3′;forward and5′AGCTTTTCCAAAAATGCCCTTGTCCGAGCTTTATCTCTTGAATAAAGCTCGGACAAGGGCAGGG-3′;reverse.
 33. A dendritic cell expressing the small interfering RNA ofclaim
 32. 34. A population of dendritic cells for use in vaccination ofa subject produced by the process of (a) obtaining dendritic cells fromthe individual; (b) causing the dendritic cells to express the antigenby either (i) exposing the dendritic cells to the antigen in cultureunder conditions promoting uptake and processing of the antigen, or (ii)transfecting the dendritic cells with a gene encoding the antigen; (c)activating the antigen-expressing dendritic cells, (d) treating thedendritic cells with an agent that inhibits MINOR expression.
 35. Apopulation of dendritic cells of claim 34 wherein the agent thatinhibits MINOR &expression is a nucleotide construct containing a smallinterfering RNA.
 36. A population of dendritic cells of claim 35 whereinthe small interfering RNA is comprised the double stranded nucleotidesequence of5′GATCCCCTGCCCTTGTCCGAGCTTTATTCAAGAGATAAAGCTCGGACAAGGGCATTTTTGGAAA-3′;forward and5′AGCTTTTCCAAAAATGCCCTTGTCCGAGCTTTATCTCTTGAATAAAGCTCGGACAAGGGCAGGG-3′;reverse.